Modified release formulations of (6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine and methods of using the same

ABSTRACT

Modified release pharmaceutical compositions (controlled release, sustained release, and/or extended release) of the R-(+) enantiomer of pramipexole (RPPX) and methods of using such compositions for the treatment of neurodegenerative diseases, or those related to mitochondrial dysfunction or increased oxidative stress are disclosed.

CROSS REFERENCE

This application claims the benefit of U.S. Provisional Application No. 60/894,799 filed Mar. 14, 2007, U.S. Provisional Application No. 60/894,829 filed Mar. 14, 2007, U.S. Provisional Application No. 60/894,814 filed Mar. 14, 2007, and U.S. Provisional Application No. 60/894,835 filed Mar. 14, 2007. This application is a continuation application of U.S. application Ser. No. 11/957,157 filed Dec. 14, 2007, which claims the benefit of U.S. Provisional Application No. 60/870,009 filed Dec. 14, 2006, and U.S. Provisional Application No. 60/979,049, filed Oct. 10, 2007, each of which is incorporated herein by reference in its entirety.

GOVERNMENT INTERESTS

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PARTIES TO A JOINT RESEARCH AGREEMENT

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INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC

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BACKGROUND

The compound 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole is a synthetic aminobenzothiazole with a chiral carbon at the 6 position and that exists as a pair of optical enantiomers depending on whether the 6-propylamino group is in the S(−) or R(+) orientation. The S(−) enantiomer, (6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine (PPX), commonly known by its USAN name, pramipexole, and commercially available as the dihydrochloride salt as Mirapex®, is a potent dopamine agonist, and thus, mimics the effects of the neurotransmitter dopamine. Therefore, pramipexole is indicated for treating Parkinson's disease, cluster headaches, restless legs syndrome and bipolar disorder with only small daily doses required and tolerated by patients activates. While pramipexole is clinically effective as a dopamine agonist, the R(+) enantiomer, (6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine (RPPX), has greatly reduced affinity for dopamine receptors and is not a clinically useful dopamine agonist. Both enantiomers, however, have been shown to exert neuroprotective activity that is independent of dopamine receptor interaction. Each is able to confer clinical neuroprotection in patients by accumulating in the cells of the brain and the spinal cord and, more specifically, in the mitochondria of these cells where they exert a dopamine independent effect on neurological function, presumably through inhibition of lipid peroxidation, normalization of mitochondrial metabolism and/or detoxification of oxygen radicals. As such, these compounds may have utility as inhibitors of the cell death cascades and loss of cell viability observed in neurodegenerative diseases.

Neuroprotective properties of PPX have been recognized as potentially useful for the treatment of neurodegenerative disorders, but clinical experience with the drug for treatment of dopamine deficiency disorders, such as PD, have shown that dosing is limited both temporally, by the need for prolonged dose titration, and absolutely, in terms of maximum tolerated dose (MTD), due to dopamine agonist-related side effects. These dosing limitations are typical for dopamine receptor agonists of this class.

The maximum allowable single starting dose for Mirapex® (an immediate release composition) is 0.125 mg, given three times a day (t.i.d.); and the maximum allowable dose for Mirapex is 1.5 mg t.i.d., providing a maximum daily dose of 4.5 mg of Mirapex® after 7-8 weeks of titration.

While these dose levels of Mirapex® are useful for treatment of the signs and symptoms of PD and RLS, in neuroprotective assays the potency of PPX as a neuroprotective is approximately 1000-fold lower than its potency as a dopamine agonist. This suggests the therapeutically useful neuroprotective doses cannot be reached using this enantiomer.

RPPX possesses similar neuroprotective potency, but lower affinity for dopamine receptors. Accordingly, it has been advanced as a potentially more useful compound for treatment of neurodegenerative disorders. However, previously reported dopamine receptor affinity difference for the RPPX compared to PPX would still impose clinically important dose limitations and would still require dose-titration and dose-limitations to avoid dopamine-related side effects. In previous reports utilizing RPPX in (ALS), dosing of RPPX was suggested to be limited and to require significant dose-titration in animal experiments. The assumed requirement for dose-titration—specifically, the requirement to start dosing at very low doses and increase the dose to a final therapeutically effective dose level over 7-8 weeks—severely limits the usefulness of the neuroprotective potential of the RPPX enantiomer Additionally, the assumed MTD would severely limit the timely exploitation of the neuroprotective potential of the RPPX enantiomer for both acute and chronic neurodegenerative disorders.

BRIEF SUMMARY OF THE INVENTION

Embodiments of the present invention are generally in the field of modified release formulations of pramipexole, particularly RPPX and pharmaceutically acceptable salts thereof. Such modified release formulations may be useful in treating neurodegenerative disorders.

The present invention unlocks the therapeutic potential of RPPX by achieving clinically purified RPPX and determining the actual in vitro and in vivo binding affinity and tolerance of a patient to purified RPPX. In accordance with embodiments of the present invention, larger doses of RPPX can be administered to a patient in need thereof.

The present invention further provides a method of treating neurodegenerative disease in a patient in need thereof, comprising administering to the patient a daily dose amount of about 25 mgs to about 5,000 mgs of RPPX, more preferably about 500 mgs to about 2,100, most preferably above 500 mgs and less than 2,100 mgs of RPPX on a daily basis, preferably in a modified release formulation of RPPX.

In some embodiments, the disease to be treated is acute and in others it is chronic. In some embodiments, the chronic neurodegenerative disease is selected from primary neurodegenerative disease, Huntington's Chorea, metabolically induced neurological damage, senile dementia of Alzheimer's type, age associated cognitive dysfunction, vascular dementia, multi-infarct dementia, Lewy body dementia, neurodegenerative dementia, neurodegenerative movement disorder, ataxia, Friedreich's ataxia, multiple sclerosis, spinal muscular atrophy, primary lateral sclerosis, seizure disorders, motor neuron disorder or disease, inflammatory demyelinating disorder, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, hepatic encephalopathy, and chronic encephalitis. In some embodiments, the chronic neurodegenerative disease is amyotrophic lateral sclerosis. In some embodiments, the patient is a naïve patient.

Modified release compositions containing RPPX are described herein. The compositions can be formulated to comprise the R(+) enantiomer of pramipexole substantially free of the S(−) enantiomer. The modified release formulations may provide sustained plasma levels of pramipexole over an extended time period. Substantially free of PPX, as used herein, refers to a pramipexole formulation wherein greater than 99.5%, greater than 99.6%, greater than 99.7%, greater than 99.8%, greater than 99.9%, preferably greater than 99.95%, and more preferably greater than 99.99% of the pramipexole molecules are RPPX, or the R(+) enantiomer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the mean plasma RPPX concentrations after oral administration of single immediate release 50 mg, 150 mg, and 300 mg doses to healthy volunteers under fasted conditions.

FIG. 2 depicts mean plasma RPPX concentrations after oral administration of single immediate release 150 mg doses to healthy volunteers under fasted and fed conditions.

FIG. 3 depicts mean plasma RPPX concentrations on Days 1 and 7 during oral administration of immediate release 50 mg and 100 mg doses on Day 1, Q12H on Days 3 through 6, and a single dose on Day 7 to healthy volunteers under fasted conditions.

FIG. 4 depicts an exposure (AUC) vs. dose (mg/m²) for male and female rats and humans (both genders).

FIG. 5 depicts mean exposure (AUC) vs. dose (mg/m²) for male and female minipigs and humans (both genders).

FIG. 6 depicts the predicted steady-state plasma concentrations of the oral administration of an exemplary 300 mg modified-release formulation once daily.

FIG. 7 depicts the predicted in vivo release profile of the oral administration of an exemplary 300 mg modified-release formulation once daily.

DETAILED DESCRIPTION

The present invention provides evidence that the dopamine receptor affinity of RPPX is actually much lower than that previously assumed, which greatly increases the clinical usefulness of the composition. It is also demonstrated herein that the functional affinity difference between the PPX and RPPX enantiomers (e.g. 10,000-20,000 fold) is much greater than previously reported. These data demonstrate that RPPX can be dosed at levels that can more fully and unexpectedly exploit the lower-potency neuroprotective potential of the compound without the theoretical limitation imposed by the assumptions about separation in dopamine receptor affinity between the enantiomers. This dosing may occur without the need for dose titration. These data also show that contamination of the composition of pure RPPX with small amounts of PPX results in dramatic shifts in the off-target activity of the composition. The application presents methods for using more chirally pure modified release compositions of RPPX in acute and chronic neurodegenerative disorders previously thought to be inaccessible to this drug, at immediate full-strength, and/or without dose-titration.

In some embodiments, the present invention provides a modified release pharmaceutical composition comprising RPPX of a sufficient dose to achieve neuroprotective, anti-oxidative, anti-apoptotic, or other beneficial cellular effects without simultaneously causing significant side effects, especially those related to dopaminergic activity. The ability to deliver clinically effective doses without dose limiting side effects is made possible by two basic discoveries: (i) the synthesis of RPPX that is pure within limits of the detection discussed herein; and (ii) the discovery that RPPX possesses substantially less affinity for dopamine receptors than previously reported. The modified release pharmaceutical composition of the present invention may be dependent, in some embodiments, on either or both the optical purity of the RPPX used in the composition and upon the limited dopaminergic activity of the chirally pure RPPX used in the composition.

Before the present compositions and methods are described, it is to be understood that this invention is not limited to the particular processes, compositions, or methodologies described, as these may vary. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. All publications mentioned herein are incorporated by reference in their entirety to the extent to support the present invention.

The compound 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole is a synthetic aminobenzothiazole derivative, having two enantiomers with the structures shown below. The (S) enantiomer is a potent agonist of the D₂ family of dopamine receptors, with particular affinity for the D₃ receptor subtype. As a dopamine agonist, PPX activates dopamine receptors, thus mimicking the effects of the neurotransmitter dopamine. The PPX stereoisomer is a potent agonist of dopamine, with only small daily doses required and indeed tolerated by patients. Both enantiomers are thought to confer neuroprotective effects by their ability to accumulate in the brain, the spinal cord and mitochondria, and by normalization of mitochondrial function and/or detoxification of oxygen radicals independent of the dopamine agonist activity, presumably through inhibition of lipid peroxidation. As such, these compounds may have utility as inhibitors of the cell death cascades and loss of cell viability observed in neurodegenerative diseases.

The degree to which dosing of a molecule has demonstrable phenotypic activity resulting from affinity to particular receptors or other pharmaco-effective proteins, even when the activity results from affinities to unknown targets, can be operationally defined in terms of whether this activity contributes in a positive way (‘on-target’ activity) or a negative way (‘off-target’ activity) to a specific and desired therapeutic effect. For any given molecule, a number of ‘off-target’ activities can theoretically be identified, but ‘on-target’ activity is restricted to the desired therapeutic effect. To the extent that these activities can be measured and quantified, or comparisons made with known standards, an index of activity can be generated for each of these categories (the ‘activity equivalent’, or AE), and one or more ratios generated to compare ‘off-target’ to ‘on-target’ activities, useful to compare potential risk-benefit ratios between molecules.

In the case of RPPX, two activities can be defined in this context. The first, which is agonist activity at a subset of human dopamine receptors and the resulting behavioral/toxicological phenotype, is ‘off-target’ activity for most neurodegenerative disorders. This activity results in dose-limiting side effects due to dopamine receptor agonist activity, and for the purposes of the current discussion can be defined to be the dopamine activity equivalent, or DAE. Throughout the application, the term “dopaminergic activity equivalent” (DAE) will be referred to which means the measure of activity at the dopamine receptors equivalent to the activity of 1 mg of PPX at the dopamine receptors. For example, a dosage of RPPX having a DAE of 0.01 would have activity at the dopamine receptors which is equivalent to the activity of 0.01 mg of PPX. The DAE can also be related to a variety of pharmaceutical terms, including maximum tolerated dose (MTD), no observable adverse effect level (NOAEL), and non-effective dose amount for the sake of clarity. For example, the NOAEL dose amount for PPX in an IMMEDIATE RELEASE is most preferably below 0.05 mg. This, in turn, corresponds to a DAE of below 0.05. A dose amount of RPPX having a DAE of 0.01 would, therefore, be below the DAE for the most preferable PPX NOAEL dose amount of 0.05 mg. In some embodiments, DAE is determined by measuring the binding affinity (IC₅₀) or activity (EC₅₀) at the D₂ and/or D₃ receptors relative to the same parameter for 1 mg of PPX. In some embodiments, DAE is determined by the binding affinity or activity at the D₂ receptor. In some embodiments, DAE is determined by the binding affinity or activity at the D₃ receptor. In some embodiments, DAE is determined by the binding affinity at the D₂ receptor. In some embodiments, DAE is determined by the binding affinity at the D₃ receptor. In some embodiments, DAE is determined by a suitable in vitro assay such as an IC₅₀ binding affinity assay for the D₂ or D₃ receptor, including those described by Schneider, C. S.; Mierau, J., “Dopamine Autoreceptor Agonists: Resolution and Pharmacological Activity of 2,6-Diaminotetrahydrobenzothiazole and an Aminothiazole Analogue of Apomorphine”, (1987). J. Med. Chem. 30:494-498; or Wong, S. K.-F.; Shrikhande, A. V., S. K.-F. Wong, “Activation of Extracellular Signal-Regulated Kinase by Dopamine D2 and D3 Receptors”, (2003) Society for Neuroscience Abstracts. This ‘off-target’ activity for RPPX in neurodegenerative disorders (other than Parkinson's disease) would be the ‘on-target’ activity for its enantiomer PPX, used to treat PD and restless legs syndrome.

Our studies suggest that the DAE for RPPX is much lower than may have been previously appreciated. For example, our studies have shown that the binding affinity for RPPX to the D₂ and D₃ dopamine receptors is about 290 and 649 times lower than PPX, respectively, when using high chiral purity RPPX. By comparison, the literature reports that the binding affinity for the RPPX to the D₂ dopamine receptor is about 9-21 times lower than PPX, while the binding affinity for the RPPX to the D₃ dopamine receptor is about 50 times lower than PPX.

Even more striking, our studies in beagle dogs indicate that the MTD dose ratio of RPPX to PPX is 10,000 while the NOAEL dose ratio of RPPX to PPX is 20,000. As a biological assay, the MTD and NOAEL in dogs reveal in vivo tolerance heretofore entirely unpredictable. Because of limitations on standard and quantitative analysis, the in vivo MTD and NOAEL in dogs may actually suggest even the slightest impurity of 0.005% could, in fact be responsible for the dopamine agonist-related side effects. These comparative studies suggest that the DAE for RPPX is much lower than may previously been appreciated.

The other activity of RPPX and PPX is neuroprotection. Neuroprotection is a phenomenon independent of mechanism, and hence qualifies as a category of activity. This ‘on-target’ activity of RPPX for the treatment of neurodegenerative disorders is measurable and approximately equivalent in both enantiomers, and can be defined in relative terms as the neuroprotective activity equivalent or NAE. The neuroprotective activity equivalent (NAE) refers to the neuroprotective activity inherent in 1 mg of PPX. Unlike the DAE, NAE has been shown to be equal in both enantiomers in a number of in vitro tests. In this example, the DAE is seen as a unit measure of the potential for adverse effects, while the NAE is seen as a unit measure of the potential for therapeutic benefit. For this example, the NAEs of both RPPX and Mirapex® may be determined from the concentrations needed to produce neuroprotection in in vitro assays.

In some embodiments, NAE can be determined by measuring the neuroprotective activity in a standard in vitro neuroprotective assay relative to the activity of 1 mg of PPX. In some embodiments, the neuroprotective activity is determined by measuring cell death in the presence of MPP+ and/or rotenone in dopaminergic and/or non-dopaminergic cells (as a non-limiting example, see the assay in M. Cu, Journal of Neurochemistry, 91:1075-1081 (2004)).

A preferred intent of the present invention is to maximize the NAE's delivered to a patient, while minimizing the number of activity equivalents suggestive of adverse events, in this case the DAE.

PPX has a high DAE/NAE ratio, due to the high dopamine affinity, while the corresponding ratio for RPPX is significantly lower. In practical terms the embodiments of the present invention provide significantly greater NAE levels and greater NAE/DAE levels than previously could have been predicted, maximizing the probability that a therapeutically effective dose amount of the neuroprotectant can be administered to a patient in need. The NAE and the DAE may be useful in terms of a ratio, particularly as a ratio of beneficial to adverse effects, and useful to define a range over which a particular composition may be administered.

Dosages of PPX, however, are limited by the dopaminergic activity of the (S) enantiomer, which can lead to adverse side effects at dosages above the “No Observable Adverse Effect Level” (NOAEL dose amount). A NOAEL dose as used herein refers to an amount of active compound or pharmaceutical agent that produces no statistically or biologically significant increases in the frequency or severity of adverse effects between an exposed population and its appropriate control; some effects may be produced at this level, but they are not considered as adverse, or as precursors to adverse effects. The exposed population may be a system, tissue, animal, individual or human that is being treated by a researcher, veterinarian, medical doctor or other clinician. With respect to PPX, exemplary adverse events are dizziness, hallucination, nausea, hypotension, somnolence, constipation, headache, tremor, back pain, postural hypotension, hypertonia, depression, abdominal pain, anxiety, dyspepsia, flatulence, diarrhea, rash, ataxia, dry mouth, extrapyramidal syndrome, leg cramps, twitching, pharyngitis, sinusitis, sweating, rhinitis, urinary tract infection, vasodilatation, flu syndrome, increased saliva, tooth disease, dyspnea, increased cough, gait abnormalities, urinary frequency, vomiting, allergic reaction, hypertension, pruritis, hypokinesia, nervousness, dream abnormalities, chest pain, neck pain, paresthesia, tachycardia, vertigo, voice alteration, conjunctivitis, paralysis, tinnitus, lacrimation, mydriasis and diplopia.

For example, a immediate release dose of 1.5 mg of PPX has been shown to cause somnolence in human subjects (Public Statement on Mirapex®, Sudden Onset of Sleep from the European Agency for the Evaluation of Medicinal Products; Boehringer Ingelheim product insert for Mirapex® which indicates that the drug is administered as three doses per day). Further, studies performed in dogs, as presented herein, (see Examples and results shown in Table 11) indicate that the NOAEL immediate release dose may be as low as 0.00125 mg/kg, which is equivalent to a human dose of 0.0007 mg/kg or 0.05 mg for a 70 kg individual. Thus, with reference to PPX, a NOAEL immediate release dose amount may be an amount below 1.5 mg, below 0.50 mg, or more preferably below 0.05 mg. With reference to DAE as defined herein, a NOAEL dose may have a DAE of below 1.5, below 0.5, or more preferably below 0.05. With respect to a modified release composition, a NOAEL dose amount may be an amount below 3.0 mg, below 1.5 mg or more preferably below 0.15 mg.

Generally, an amount larger than the non-effective dose amount of PPX is necessary to have a therapeutic effect in treating diseases alleviated by dopamine agonist activity. This amount, however, may not be desired when a neuroprotective effect is sought as it may lead to the described adverse side effects. A “non-effective dose amount” as used herein refers to an amount of active compound or pharmaceutical agent that elicits a biological or medicinal response similar to the biological or medicinal response of a placebo as observed in a tissue, system, animal, individual or human that is being treated by a researcher, veterinarian, medical doctor or other clinician. A “non-effective dose amount” may therefore elicit no discernable difference from placebo in positive effects as observed in a tissue, system, animal, individual or human that is being treated by a researcher, veterinarian, medical doctor or other clinician. As such, the “non-effective dose amount” is not expected to (1) prevent a disease; for example, preventing a disease, condition or disorder in an individual that may be predisposed to the disease, condition or disorder but does not yet experience or display the pathology or symptomatology of the disease; (2) inhibit the disease; for example, inhibiting a disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., arresting or slowing further development of the pathology and/or symptomatology), or (3) ameliorate the disease; for example, ameliorating a disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., reversing or reducing the pathology and/or symptomatology).

As an example, in monkeys treated with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), a known dopaminergic neurotoxin, PPX has been shown to antagonize motor deficits and Parkinson-like symptoms in a dose-dependent manner, with the lowest effective oral immediate release dose being 0.053 mg/kg (see Scientific Discussion at http://www.emea.europa.eu/humandocs/PDFs/EPAR/Sifrol/059197EN6.pdf). This would be equivalent to a human immediate release dose of 0.017 mg/kg, or 1.2 mg for a 70 kg individual. In human trials, the lowest effective oral immediate release dose of PPX with a significant effect versus placebo in the treatment of Parkinson's disease was found to be 1.1 mg/day. Individual patients may need doses higher than 1.1 mg/day to gain a sufficient effect above the placebo effect (Initial Scientific Discussion for the Approval of Mirapex from the European Agency for the Evaluation of Medicinal Products). In human trials, the lowest effective dose with a significant effect versus placebo in the treatment of restless legs syndrome was found to be 0.25 mg/day (Boehringer Ingelheim product insert for Mirapex®). Therefore, with reference to PPX, a non-effective dose amount may be an amount below 1.0 mg/day, below 0.75 mg/day, below 0.5 mg/day, below 0.25 mg/day, or preferably below 0.125 mg/day. With reference to DAE, a non-effective dose amount per day may have a DAE per day below 1.0, below 0.75, below 0.5, below 0.25, or preferably below 0.125.

Other limits on the amount of PPX which can be administered to a patient also include the maximum recommended therapeutic dose and the maximum tolerated dose. A “maximum recommended therapeutic dose” (MRTD) refers to the dosages compiled by the FDA's Center for Drug Evaluation and Research, Office of Pharmaceutical Science, Informatics and Computational Safety Analysis Staff's Maximum Recommended Therapeutic Dose and as described in Matthews, et al., “Assessment of the Health Effects of Chemicals in Humans: 1. QSAR Estimation of the Maximum Recommended Therapeutic Dose (MRTD) and No Effect Level (NOEL) of Organic Chemicals Based on Clinical Trial Data,” Current Drug Discovery Technologies, 2004, 1:61-76). The FDA's MRTD database cites a MRTD for PPX of 0.11 mg/kg/day or 7.0 mg/day for a 70 lb. person. Matthews, in turn, estimates that a NOEL (no adverse effect level) usually is about one-tenth of the MRTD, which corresponds to 0.01 mg/kg or about 0.7 mg/day for a 70 lb. person.

Because of its adverse impact on naïve patients, PPX must be titrated over the course of weeks to reach these dosages without dose limiting adverse effects (such as that documented in Boehringer Ingelheim product insert for Mirapex®). For example, for restless legs syndrome, the recommended starting daily dose amount of Mirapex®, which is an immediate release composition, is 0.125 mg taken once daily 2-3 hours before bedtime. For patients requiring additional symptomatic relief, the daily dose may be increased to 0.25 mg over 4 to 7 day period and then to 0.5 mg over a second 4 to 7 day period. For the treatment of Parkinson's disease, the package insert recommends the following titration schedule for Mirapex®:

Week Dosage (mg) Total daily dose (mg) 1 0.125 tid 0.375 2 0.25 tid 0.75 3 0.5 tid 1.5 4 0.75 tid 2.25 5 1.0 tid 3.0 6 1.25 tid 3.75 7 1.5 tid 4.5

A “maximum tolerated dose” (MTD) as used herein refers to an amount of active compound or pharmaceutical agent which elicits significant toxicity in a tissue, system, animal, individual or human that is being treated by a researcher, veterinarian, medical doctor or other clinician. Single dose toxicity of PPX after oral administration has been studied in rodents, dogs, monkeys and human. In rodents, deaths occurred at immediate release doses of 70-105 mg/kg and above (Initial Scientific Discussion for the Approval of Mirapex from the European Agency for the Evaluation of Medicinal Products). This is equivalent to a human dose of 7-12 mg/kg, or approximately 500-850 mg for a 70 kg individual. In human subjects, a starting daily dose of PPX of greater than 0.20 mg was not tolerated when administered to a naïve patient. In dogs, vomiting occurred at 0.0007 mg/kg and above while monkeys displayed major excitation at 3.5 mg/kg. Further, the product insert for Mirapex® sets the maximally tolerated dose for humans at 4.5 mg/day, administered as three 1.5 mg single dosages. However, the 4.5 mg/day dosage is not administered to a naïve patient, but instead, reached after a titration regimen (such as that documented in the product insert for Mirapex®). Generally, the starting daily dosage for administration to a naïve patient is a 0.125 mg immediate release dose administered three times per day and a seven-week titration schedule is recommended to reach a 1.5 mg dose administered three times daily. All species showed signs of toxicity related to exaggerated pharmacodynamic responses to PPX. For example, behavioral changes including hyperactivity were common and led to a number of secondary effects, such as reduced body weight and other stress-induced symptoms. In minipigs and monkeys, PPX moderately affected cardiovascular parameters. In rats, the potent prolactin-inhibitory effect of pramipexole affected reproductive organs (e.g. enlarged corpora lutea, pyometra), and showed a dose-related retinal degeneration during long-term exposure (Initial Scientific Discussion for the Approval of Mirapex from the European Agency for the Evaluation of Medicinal Products). Studies in dogs indicate a MTD amount of PPX for a human subject may be an amount below 4.5 mg/day, preferably below 1.5 mg/day. Further, the MID amount for a human subject may be an amount below 0.3 mg/immediate release dose based on results of studies disclosed herein, and preferably below 0.2 mg/immediate release dose (see Table 11). With reference to DAE, the MTD amount may have a DAE of below 1.5, 0.9 and 0.6.

Given the limits on the amount of PPX that can be administered to a patient, the use of the embodiments of the present invention presents a clinically important alternative for the development of new neuroprotective therapies. The literature previously reported that the binding affinity of RPPX at the D₂ receptor was approximately 9 to 21 times less than about that of PPX, while the binding affinity of RPPX at the D₃ receptor was approximately 50 times less than about that of PPX (Table 10). These literature-derived comparative binding affinity ratios suggest that RPPX can be administered only at somewhat higher dosages than PPX. This limitation may occur because the exquisite sensitivity of tissues, systems, animals, and human subjects to the effects of dopamine agonism would preclude the use of RPPX at doses that exceed tolerated doses of PPX by a factor greater than the literature derived comparative binding affinity ratios of the two enantiomers.

The seeming preclusion of higher doses of RPPX can be demonstrated by reference to a theoretical 50 mg ER tablet. Assuming a 9 times difference in binding affinities, a 50 mg ER tablet which is 99.95% pure would have approximately 5.575 DAE (5.55 DAE from the RPPX and 0.025 DAE from the PPX). Similarly, a 25 mg tablet would be expected to exhibit a DAE of 2.79 (2.78 from the RPPX and 0.0125 DAE from the PPX). The MTD of PPX after a seven week titration regimen is 4.5 mg, or 1.5 mg three times a day, which is equivalent to a 4.5 DAE in a day. Further, the NOAEL dose amount for PPX is below 4.5 mg, preferably below 1.50 mg, or more preferably below 0.15 mg, which are each equivalent to DAE, DAE, and DAE, respectively. Given that the daily MTD for PPX has 4.5 DAE and the NOAEL of PPX has less than about 4.5 DAE, a ER dosage of 50 mg with a DAE of 5.55 when referring solely to the literature derived comparative binding affinity ratios. Further, use of a high chiral purity of 99.95% as used in these theoretical dosages, would result in unacceptably high DAE of 5.55 the daily dosage MTD DAE of 4.5 mg, and far beyond the preferable NOAELs of 1.5 DAE and 0.15 DAE.

To the contrary, in some embodiments, an aspect of the present invention involves unexpectedly high chiral purities that have been attained. These purities have led to MTDs or NOAELs for RPPX which are higher than previously appreciated based on the literature derived comparative binding affinities. In some embodiments, the present invention provides modified release pharmaceutical compositions, starting doses, method of treatment, and kits comprising RPPX of high chiral purity. Pursuant the discussion above, a 50 mg modified release dosage with a similar chiral purity of 99.95% would be predicted to be well above the MTD or NOAEL for PPX and, therefore, result in observable adverse side effects. Studies in dogs, however, suggest that the high chiral purity RPPX results in NOAEL dose amounts unexpectedly higher than those that may have been appreciated (Table 10). Incredibly, a 25 mg/kg immediate release dosage of RPPX with no detectable amount of PPX (0.05% limit of detection) resulted in no observable effects in dogs, which is unexpected based on the literature binding affinity data.

Further, the studies in dogs demonstrate the high (approaching absolute) chiral purity of the compositions for the (R)-enantiomer. RPPX is administered in high dose levels in the studies disclosed herein (equivalent to human doses of 1,000 mg to 3,000 mg; see Examples), so that even the smallest amount of PPX would contribute to the observed NOAEL and MTD. For example, with reference to human equivalence doses based on data obtained in dogs, the MTD for the (R)-enantiomer has been shown to be equivalent to about 3,000 mg for a 70 kg human subject, while the equivalent MTD for the (S)-enantiomer would be equivalent to only 0.30 mg for that same subject (Table 11). That is a difference of 10,000-fold. The NOAEL dose for the (R)-enantiomer is 20,000-fold greater than for the (S)-enantiomer (Table 11). Thus, the RPPX compositions used in these studies must be at least 99.99% pure if one were to assume that the observed side effects stemmed only from contamination by the (S)-enantiomer. On the other hand, these data demonstrate the high dose levels of the (R)-enantiomer of pramipexole that may be administered safely. These data highlight the usefulness of the high chiral purity for RPPX in various embodiments of the present invention.

The present invention further provides modified release pharmaceutical compositions, starting doses, methods, and kits comprising RPPX with higher dosages and higher chiral purities. As discussed above, the literature previously suggested that the comparative binding affinity ratios at the D₂ and D₃ receptors were approximately 9 to 21, and 50, respectively (See Example 1 and Table 10 below). It has been unexpectedly found that the comparative binding affinity ratios of PPX:RPPX at the D₂ and D₃ receptors are approximately 290 and 649, when using high chiral purity RPPX.

As is discussed in greater detail below this suggests that the comparative binding affinity ratios are about 13 to 32 times the comparative binding affinity ratios reported in the literature. The literature is replete with discussion of the adverse impact of PPX. Although the in vitro data and binding affinity is presented in support of the present invention is compelling, the importance of an economical and efficient synthesis becomes apparent when comparing the in vitro and in vivo data presented herein. The in vivo clinical observations in beagle dogs indicate that the MTD dose ratio of RPPX to PPX is 10,000, while the NOAEL dose ratio of RPPX to PPX is 20,000. The absolute MTD dose ratio may be higher because the chiral purities reported herein are limited to level of detection (See Example 2 and Table 11). Based on the chiral purity and the in vitro comparative binding affinity ratios, clinical NOAEL dose ratios, or clinical MTD dose ratios (herein “comparative ratios”), it is now possible to predict the DAE for a given dosage of RPPX. Table 1 shows the DAE for a 25 mg ER dose of RPPX as a function of comparative ratio and chiral purity. These data show that a much lower DAIS can unexpectedly result from a 25 mg ER dosage form of RPPX than may have been previously appreciated, due to the lower comparative ratios described herein when compared to the literature derived comparative ratios.

TABLE 1 DAE for a 25 mg ER dose of RPPX as a function of % chiral purity and the comparative ratio Percent Chiral 20,000 10,000 5,000 2,400 100 10 Purity for comparative comparative comparative comparative comparative comparative RPPX ratio ratio ratio ratio ratio ratio 99.9967 0.0020749 0.0033249 0.0058248 0.0112413 0.2508168 2.5007425 99.9958 0.0022999 0.0035498 0.0060498 0.0114662 0.2510395 2.5009450 99.9950 0.0024999 0.0037499 0.0062498 0.0116661 0.2512375 2.5011250 99.9933 0.0029249 0.0041783 0.0066747 0.0120909 0.2516583 2.5015075 99.9900 0.0037499 0.0049998 0.0074995 0.0129156 0.2524750 2.5022500 99.9833 0.0054248 0.0066746 0.0091742 0.0145899 0.2531333 2.5037575 99.9800 0.0062498 0.0074995 0.0099990 0.0154158 0.2549500 2.5045000 99.9750 0.0074997 0.0087494 0.0112488 0.0166641 0.2561875 2.5056250 99.9667 0.0095746 0.0108242 0.0133233 0.0187382 0.2582418 2.5074925 99.9583 0.0116745 0.0129239 0.0154229 0.0208373 0.2603208 2.5093825 99.9500 0.0137494 0.0149988 0.0174975 0.0229115 0.2623750 2.5112500 99.9333 0.0179242 0.0191733 0.0216717 0.0270847 0.2665083 2.5150075 99.9000 0.0262488 0.0274975 0.0299950 0.0354063 0.2747500 2.5225000 99.8333 0.0429229 0.0441798 0.0466666 0.0520743 0.2912583 2.5375075 99.8000 0.0512475 0.0524950 0.0549900 0.0603958 0.2995000 2.5450000 99.7500 0.0637469 0.0649938 0.0674875 0.0728906 0.3118750 2.5562500 99.6667 0.0845708 0.0858167 0.0883093 0.0937065 0.3324918 2.5749925 99.5800 0.1062448 0.1074895 0.1099790 0.1153729 0.3539500 2.5945000 99.5000 0.1262438 0.1274875 0.1299750 0.1353656 0.3737500 2.6125000 99.3333 0.1679167 0.1691583 0.1764167 0.1770222 0.4150083 2.6500075 99.0000 0.2512375 0.2524750 0.2549500 0.2603125 0.4975000 2.7250000 98.3300 0.4187291 0.4199583 0.4224165 0.4277427 0.6633250 2.8757500 98.0000 0.5102250 0.5024500 0.5049000 0.5102083 0.7450000 2.9500000 97.5000 0.62621875 0.6274375 0.629875 0.6351563 0.86875 3.0625

Table 1 attempts to illustrate the importance of both purity and affinity on even a 25 mg single oral dosage. Assumptions regarding dopaminergic activity of the RPPX at the dopamine receptors would seemingly preclude even a high purity (even 100% pure) 25 mg RPPX tablet. Based upon the disclosure of the present invention one can immediately envisage numerous tables to illustrate the point. Tables 1A and 1B below are intended to illustrate the importance of purity for a single oral dosage form of RPPX by illustrating the impact of even the smallest contamination of the composition by PPX

TABLE 1A “NOAEL” dosages of RPPX compositions (based on DAE <0.15) 50 mg 100 mg 150 mg 200 mg 250 mg 500 mg (R)-purity % 99.9000 99.9500 99.9667 99.9750 99.9800 99.9900 (S)-impurity % 0.1000 0.0500 0.0333 0.0250 0.0200 0.0100 (S)-impurity 0.05 0.05 0.05 0.05 0.05 0.05 DAE

TABLE 1B “Non-effective” dosages of RPPX compositions (based on DAE <0.125) 50 mg 100 mg 150 mg 200 mg 250 mg 500 mg (R)-purity % 99.7500 99.8750 99.9170 99.9380 99.9500 99.9750 (S)-impurity % 0.2500 0.1250 0.0830 0.0620 0.0500 0.0250 (S)-impurity 0.125 0.125 0.125 0.125 0.125 0.125 DAE

No one has appreciated or articulated that synthetic methodology was necessary to achieve purities that exceed typical detection limits. Further, no one has suggested this single dosage must be 99.95% or greater in purity to be suitable for its intended purpose.

Based on the comparative ratios for binding affinity, NOAEL and MTD values, it is then possible to predict the amount of RPPX that could be administered which would be equivalent to a non-effective dose amount of the PPX. Table 2 shows DAE as a function of a dosage of RPPX (left hand column) and the comparative ratio (top row). With reference to Table 2, a unit dose can be chosen which allows for an amount of RPPX having DAE which is equal to the non-effective amount of PPX. Indeed, unless a dual DAE/NAE effect is desired, a DAE would be avoided or minimized in a pharmaceutical composition. Thus, any ER dose greater than 50 milligrams would not be expected to avoid off-target activity and would be expressly avoided by one skilled in the art. This is not true if, as in present invention, the comparative ratios exceed 200. This is best illustrated by Table 2.

TABLE 2 DAE as a function of RPPX and comparative ratio (assuming 100% chiral purity of RPPX) 20,000 10,000 5,000 2,400 1,700 1,300 650 400 300 200 100 50 2.5 0.00013 0.00025 0.0005 0.0010 0.0015 0.0019 0.0038 0.0063 0.0083 0.0125 0.0250 0.0500 5 0.00025 0.00050 0.001 0.0021 0.0029 0.0038 0.0077 0.0125 0.0167 0.0250 0.0500 0.1000 6.25 0.00031 0.00063 0.00125 0.0026 0.0037 0.0048 0.0096 0.0156 0.0208 0.0313 0.0625 0.1250 10 0.00050 0.00100 0.002 0.0042 0.0059 0.0077 0.0154 0.0250 0.0333 0.0500 0.1000 0.2000 12.5 0.00063 0.00125 0.0025 0.0052 0.0074 0.0096 0.0192 0.0313 0.0417 0.0625 0.1250 0.2500 15 0.00075 0.00150 0.003 0.0063 0.0088 0.0115 0.0231 0.0375 0.0500 0.0750 0.1500 0.3000 20 0.00100 0.00200 0.004 0.0083 0.0118 0.0154 0.0308 0.0500 0.0667 0.1000 0.2000 0.4000 25 0.00125 0.00250 0.005 0.0104 0.0147 0.0192 0.0385 0.0625 0.0833 0.1250 0.2500 0.5000 32.5 0.00163 0.00325 0.0065 0.0135 0.0191 0.0250 0.0500 0.0813 0.1083 0.1625 0.3250 0.6500 37.5 0.00188 0.00375 0.0075 0.0156 0.0221 0.0288 0.0577 0.0938 0.1250 0.1875 0.3750 0.7500 50 0.0025 0.0050 0.0100 0.0208 0.0294 0.0385 0.0769 0.1250 0.1667 0.2500 0.5000 1.0000 65 0.0033 0.0065 0.0130 0.0271 0.0382 0.0500 0.1000 0.1625 0.2167 0.3250 0.6500 1.3000 80 0.0040 0.0080 0.0160 0.0333 0.0471 0.0615 0.1231 0.2000 0.2667 0.4000 0.8000 1.6000 81.25 0.0041 0.0081 0.0163 0.0339 0.0478 0.0625 0.1250 0.2031 0.2708 0.4063 0.8125 1.6250 85 0.0043 0.0085 0.0170 0.0354 0.0500 0.0654 0.1308 0.213 0.283 0.425 0.850 1.700 100 0.0050 0.0100 0.0200 0.0417 0.0588 0.0769 0.1538 0.250 0.333 0.500 1.000 2.000 120 0.0060 0.0120 0.0240 0.0500 0.0706 0.0923 0.1846 0.300 0.400 0.600 1.200 2.400 130 0.0065 0.0130 0.0260 0.0542 0.0765 0.1000 0.2000 0.325 0.433 0.650 1.300 2.600 150 0.0075 0.0150 0.0300 0.0625 0.0882 0.1154 0.2308 0.375 0.500 0.750 1.500 3.000 162.5 0.0081 0.0163 0.0325 0.0677 0.0956 0.1250 0.2500 0.406 0.542 0.813 1.625 3.250 200 0.0100 0.0200 0.0400 0.0833 0.1176 0.1538 0.3077 0.500 0.667 1.000 2.000 4.000 212.5 0.011 0.021 0.043 0.0885 0.1250 0.1635 0.3269 0.531 0.708 1.063 2.125 4.250 250 0.013 0.025 0.050 0.1042 0.1471 0.1923 0.3846 0.625 0.833 1.250 2.500 5.000 260 0.013 0.026 0.052 0.1083 0.1529 0.2000 0.4000 0.650 0.867 1.300 2.600 5.200 300 0.015 0.030 0.060 0.1250 0.1765 0.2308 0.4615 0.750 1.000 1.500 3.000 6.000 325 0.016 0.033 0.065 0.135 0.191 0.250 0.500 0.813 1.083 1.625 3.250 6.500 340 0.017 0.034 0.068 0.142 0.200 0.262 0.523 0.850 1.133 1.700 3.400 6.800 350 0.018 0.035 0.070 0.146 0.206 0.269 0.538 0.875 1.167 1.750 3.500 7.000 400 0.020 0.040 0.080 0.167 0.235 0.308 0.615 1.000 1.333 2.000 4.000 8.000 500 0.025 0.050 0.100 0.208 0.294 0.385 0.769 1.3 1.7 2.5 5.0 10.0 600 0.030 0.060 0.120 0.250 0.353 0.462 0.923 1.5 2.0 3.0 6.0 12.0 625 0.031 0.063 0.125 0.260 0.368 0.481 0.962 1.6 2.1 3.1 6.3 12.5 650 0.033 0.065 0.130 0.271 0.382 0.500 1.000 1.6 2.2 3.3 6.5 13.0 850 0.043 0.085 0.170 0.354 0.500 0.654 1.308 2.1 2.8 4.3 8.5 17.0 1000 0.050 0.100 0.200 0.417 0.588 0.769 1.538 2.5 3.3 5.0 10.0 20.0 1200 0.060 0.120 0.240 0.500 0.706 0.923 1.846 3.0 4.0 6.0 12.0 24.0 1250 0.063 0.125 0.250 0.521 0.735 0.962 1.923 3.1 4.2 6.3 12.5 25.0 1300 0.065 0.130 0.260 0.542 0.765 1.000 2.000 3.3 4.3 6.5 13.0 26.0 1700 0.085 0.170 0.340 0.708 1.000 1.308 2.615 4.3 5.7 8.5 17.0 34.0 2000 0.100 0.200 0.400 0.833 1.176 1.538 3.077 5.0 6.7 10.0 20.0 40.0 2400 0.120 0.240 0.480 1.00 1.41 1.85 3.69 6.0 8.0 12.0 24.0 48.0 2500 0.125 0.250 0.500 1.04 1.47 1.92 3.85 6.3 8.3 12.5 25.0 50.0 3250 0.163 0.325 0.650 1.35 1.91 2.50 5.00 8.1 10.8 16.3 32.5 65.0 5000 0.250 0.500 1.000 2.08 2.94 3.85 7.69 12.5 16.7 25.0 50.0 100.0 6500 0.325 0.650 1.300 2.71 3.82 5.00 10.00 16.3 21.7 32.5 65.0 130.0 8500 0.425 0.850 1.700 3.54 5.00 6.54 13.08 21.3 28.3 42.5 85.0 170.0 10000 0.500 1.000 2.000 4.17 5.88 7.69 15.38 25.0 33.3 50.0 100.0 200.0 20000 1.000 2.000 4.000 8.33 11.76 15.38 30.77 50.0 66.7 100.0 200.0 400.0 A DAE equivalent to a preferred non-effective dose amount of the PPX may be below 1.0 mg; more preferably below 0.5 mg, and more preferably below 0.125 mg.

Similarly, one can ascertain the amount of RPPX that could be administered which would be equivalent to a no observable adverse effect level dose amount of the PPX. Table 3 shows DAE as a function of a dosage of RPPX (left hand column) and the comparative ratio (top row). With reference to Table 3, a unit dose can be chosen which allows for an amount of RPPX having a DAE equal to the NOAEL dose amount of PPX. While 0.125 avoids unwanted effects, less than 0.15 avoids NOAEL. The difference in literature report and actual results is even more striking in Table 3.

TABLE 3 DAE as a function of dosages of RPPX and comparative ratio (assuming 100% chiral purity of RPPX) 20,000 10,000 5,000 2,400 1,700 1,300 650 400 300 200 100 50.000 2.5 0.00013 0.00025 0.00050 0.00104 0.0015 0.0019 0.0038 0.0063 0.0083 0.0125 0.025 0.050 5 0.00025 0.00050 0.00100 0.00208 0.0029 0.0038 0.0077 0.0125 0.0167 0.0250 0.050 0.100 6.25 0.00031 0.00063 0.00125 0.00260 0.0037 0.0048 0.0096 0.0156 0.0208 0.0313 0.063 0.125 10 0.00050 0.00100 0.00200 0.00417 0.0059 0.0077 0.0154 0.0250 0.0333 0.0500 0.100 0.200 12.5 0.00063 0.00125 0.00250 0.00521 0.0074 0.0096 0.0192 0.0313 0.0417 0.0625 0.125 0.250 15 0.00075 0.00150 0.00300 0.00625 0.0088 0.0115 0.0231 0.0375 0.0500 0.0750 0.150 0.300 20 0.00100 0.00200 0.00400 0.00833 0.0118 0.0154 0.0308 0.0500 0.0667 0.1000 0.200 0.400 25 0.00125 0.00250 0.00500 0.01042 0.0147 0.0192 0.0385 0.0625 0.0833 0.1250 0.250 0.500 32.5 0.00163 0.00325 0.00650 0.01354 0.0191 0.0250 0.0500 0.0813 0.1083 0.1625 0.325 0.650 37.5 0.00188 0.00375 0.00750 0.01563 0.0221 0.0288 0.0577 0.0938 0.1250 0.1875 0.375 0.750 50 0.0025 0.0050 0.0100 0.0208 0.0294 0.0385 0.0769 0.1250 0.1667 0.2500 0.500 1.000 65 0.0033 0.0065 0.0130 0.0271 0.0382 0.0500 0.1000 0.1625 0.2167 0.3250 0.650 1.300 80 0.0040 0.0080 0.0160 0.0333 0.0471 0.0615 0.1231 0.2000 0.2667 0.4000 0.800 1.600 81.25 0.0041 0.0081 0.0163 0.0339 0.0478 0.0625 0.1250 0.2031 0.2708 0.4063 0.813 1.625 85 0.0043 0.0085 0.0170 0.0354 0.0500 0.0654 0.1308 0.2125 0.2833 0.4250 0.850 1.700 100 0.0050 0.0100 0.0200 0.0417 0.0588 0.0769 0.1538 0.2500 0.3333 0.5000 1.000 2.000 120 0.0060 0.0120 0.0240 0.0500 0.0706 0.0923 0.1846 0.3000 0.4000 0.6000 1.200 2.400 130 0.0065 0.0130 0.0260 0.0542 0.0765 0.1000 0.2000 0.3250 0.4333 0.6500 1.300 2.600 150 0.0075 0.0150 0.0300 0.0625 0.0882 0.1154 0.2308 0.3750 0.5000 0.7500 1.500 3.000 162.5 0.0081 0.0163 0.0325 0.0677 0.0956 0.1250 0.2500 0.4063 0.5417 0.8125 1.625 3.250 200 0.0100 0.0200 0.0400 0.0833 0.1176 0.1538 0.3077 0.5000 0.6667 1.0000 2.000 4.000 212.5 0.0106 0.0213 0.0425 0.0885 0.1250 0.1635 0.3269 0.5313 0.7083 1.0625 2.125 4.250 250 0.0125 0.0250 0.0500 0.1042 0.1471 0.1923 0.3846 0.6250 0.8333 1.2500 2.500 5.000 260 0.0130 0.0260 0.0520 0.1083 0.1529 0.2000 0.4000 0.6500 0.8667 1.3000 2.600 5.200 300 0.0150 0.0300 0.0600 0.1250 0.1765 0.2308 0.4615 0.7500 1.0000 1.5000 3.000 6.000 400 0.0200 0.0400 0.0800 0.1667 0.2353 0.3077 0.6154 1.0000 1.3333 2.0000 4.000 8.000 500 0.0250 0.0500 0.1000 0.2083 0.2941 0.3846 0.7692 1.2500 1.6667 2.5000 5.000 10.000 600 0.0300 0.0600 0.1200 0.2500 0.3529 0.4615 0.9231 1.5000 2.0000 3.0000 6.000 12.000 625 0.0313 0.0625 0.1250 0.2604 0.3676 0.4808 0.9615 1.5625 2.0833 3.1250 6.250 12.500 650 0.0325 0.0650 0.1300 0.2708 0.3824 0.5000 1.0 1.6 2.2 3.3 6.5 13.0 850 0.0425 0.0850 0.1700 0.3542 0.5000 0.6538 1.3 2.1 2.8 4.3 8.5 17.0 1000 0.0500 0.1000 0.2000 0.4167 0.5882 0.7692 1.5 2.5 3.3 5.0 10.0 20.0 1200 0.0600 0.1200 0.2400 0.5000 0.7059 0.9231 1.8 3.0 4.0 6.0 12.0 24.0 1250 0.0625 0.1250 0.2500 0.5208 0.7353 0.9615 1.9 3.1 4.2 6.3 12.5 25.0 2000 0.1000 0.2000 0.4000 0.8333 s 1.5385 3.1 5.0 6.7 10.0 20.0 40.0 2400 0.1200 0.2400 0.4800 1.0000 1.4118 1.8462 3.7 6.0 8.0 12.0 24.0 48.0 2500 0.1250 0.2500 0.5000 1.0417 1.4706 1.9231 3.8 6.3 8.3 12.5 25.0 50.0 3250 0.1625 0.3250 0.6500 1.3542 1.9118 2.5000 5.0 8.1 10.8 16.3 32.5 65.0 5000 0.2500 0.5000 1.00 2.08 2.94 3.85 7.7 12.5 16.7 25.0 50.0 100.0 6500 0.3250 0.6500 1.30 2.71 3.82 5.00 10.0 16.3 21.7 32.5 65.0 130.0 8500 0.4250 0.8500 1.70 3.54 5.00 6.54 13.1 21.3 28.3 42.5 85.0 170.0 10000 0.5000 1.0000 2.00 4.17 5.88 7.69 15.4 25.0 33.3 50.0 100.0 200.0 20000 1.0000 2.0000 4.00 8.33 11.76 15.38 30.8 50.0 66.7 100.0 200.0 400.0 A DAE equivalent to a preferred no observable adverse effect level (NOAEL) dose amount of the PPX may be below 1.5 mg, preferably below 0.15 mg.

Further, Table 4 shows DAE as a function of a dosage of RPPX (left hand column) and the comparative ratio (top row). With reference to Table 4, a unit dose can be chosen which allows a dose amount of RPPX having a particular DAE.

TABLE 4 DAE as a function of dosages of RPPX and comparative ration (assuming 100% chiral purity of RPPX) 20,000 10,000 5,000 2,400 1,700 1,300 650 400 300 200 100 50 2.5 0.00013 0.00025 0.00050 0.0010 0.0015 0.0019 0.0038 0.0063 0.0083 0.013 0.025 0.050 5 0.00025 0.00050 0.00100 0.0021 0.0029 0.0038 0.0077 0.0125 0.0167 0.025 0.050 0.100 6.25 0.00031 0.00063 0.00125 0.0026 0.0037 0.0048 0.0096 0.0156 0.0208 0.031 0.063 0.125 10 0.00050 0.00100 0.00200 0.0042 0.0059 0.0077 0.0154 0.0250 0.0333 0.050 0.100 0.200 12.5 0.00063 0.00125 0.00250 0.0052 0.0074 0.0096 0.0192 0.0313 0.0417 0.063 0.125 0.250 15 0.00075 0.00150 0.00300 0.0063 0.0088 0.0115 0.0231 0.0375 0.0500 0.075 0.150 0.300 20 0.00100 0.00200 0.00400 0.0083 0.0118 0.0154 0.0308 0.0500 0.0667 0.100 0.200 0.400 25 0.00125 0.00250 0.00500 0.0104 0.0147 0.0192 0.0365 0.0625 0.0833 0.125 0.250 0.500 40 0.00200 0.00400 0.00800 0.0167 0.0235 0.0308 0.0615 0.1000 0.1333 0.200 0.400 0.800 50 0.00250 0.00500 0.010 0.021 0.029 0.038 0.077 0.125 0.167 0.250 0.500 1.000 60 0.00300 0.00600 0.012 0.025 0.035 0.046 0.092 0.150 0.200 0.300 0.600 1.200 65 0.00325 0.00650 0.013 0.027 0.038 0.050 0.100 0.163 0.217 0.325 0.650 1.300 80 0.00400 0.00800 0.016 0.033 0.047 0.062 0.123 0.200 0.267 0.400 0.800 1.600 85 0.00425 0.00850 0.017 0.035 0.050 0.065 0.131 0.213 0.283 0.425 0.850 1.700 100 0.00500 0.010 0.020 0.042 0.059 0.077 0.154 0.250 0.333 0.500 1.000 2.000 130 0.00650 0.013 0.026 0.054 0.076 0.100 0.200 0.325 0.433 0.650 1.300 2.600 150 0.00750 0.015 0.030 0.063 0.088 0.115 0.231 0.375 0.500 0.750 1.500 3.000 162.5 0.00813 0.016 0.033 0.068 0.096 0.125 0.250 0.406 0.542 0.813 1.625 3.250 200 0.010 0.020 0.040 0.083 0.118 0.154 0.308 0.500 0.667 1.000 2.000 4.000 260 0.013 0.026 0.052 0.108 0.153 0.200 0.400 0.650 0.867 1.300 2.6 5.2 300 0.015 0.030 0.060 0.125 0.176 0.231 0.462 0.750 1.000 1.500 3.0 6.0 325 0.016 0.033 0.065 0.135 0.191 0.250 0.500 0.813 1.083 1.625 3.3 6.5 340 0.017 0.034 0.068 0.142 0.200 0.262 0.523 0.850 1.133 1.700 3.4 6.8 350 0.018 0.035 0.070 0.146 0.206 0.269 0.538 0.875 1.167 1.750 3.5 7.0 400 0.020 0.040 0.080 0.167 0.235 0.308 0.615 1.000 1.333 2.000 4.0 8.0 480 0.024 0.048 0.096 0.200 0.282 0.369 0.738 1.200 1.600 2.400 4.8 9.6 500 0.025 0.050 0.100 0.208 0.294 0.385 0.769 1.250 1.667 2.500 5.0 10.0 600 0.030 0.060 0.120 0.250 0.353 0.462 0.923 1.500 2.000 3.000 6.0 12.0 850 0.043 0.085 0.170 0.354 0.500 0.654 1.308 2.125 2.833 4.250 8.5 17.0 1000 0.050 0.100 0.200 0.417 0.588 0.769 1.538 2.500 3.3 5.0 10.0 20.0 1200 0.060 0.120 0.240 0.500 0.706 0.923 1.846 3.000 4.0 6.0 12.0 24.0 1250 0.063 0.125 0.250 0.521 0.735 0.962 1.923 3.125 4.2 6.3 12.5 25.0 1300 0.065 0.130 0.260 0.542 0.765 1.000 2.000 3.250 4.3 6.5 13.0 26.0 1500 0.075 0.150 0.300 0.625 0.882 1.154 2.308 3.750 5.0 7.5 15.0 30.0 1700 0.085 0.170 0.340 0.708 1.000 1.308 2.615 4.250 5.7 8.5 17.0 34.0 2000 0.100 0.200 0.400 0.833 1.176 1.538 3.077 5.000 6.7 10.0 20.0 40.0 2400 0.120 0.240 0.480 1.000 1.412 1.846 3.692 6.000 8.0 12.0 24.0 48.0 2500 0.125 0.250 0.500 1.042 1.471 1.923 3.846 6.250 8.3 12.5 25.0 50.0 3250 0.163 0.325 0.650 1.354 1.912 2.500 5.000 8.125 10.8 16.3 32.5 65.0 4000 0.200 0.400 0.800 1.667 2.353 3.077 6.154 10.000 13.3 20.0 40.0 80.0 5000 0.250 0.500 1.000 2.083 2.941 3.846 7.7 12.5 16.7 25.0 50.0 100.0 6500 0.325 0.650 1.300 2.708 3.824 5.000 10.0 16.3 21.7 32.5 65.0 130.0 8500 0.425 0.850 1.700 3.542 5.000 6.538 13.1 21.3 28.3 42.5 85.0 170.0 10000 0.500 1.000 2.000 4.167 5.882 7.692 15.4 25.0 33.3 50.0 100.0 200.0 12000 0.600 1.200 2.400 5.000 7.059 9.231 18.5 30.0 40.0 60.0 120.0 240.0 20000 1.000 2.000 4.000 8.333 11.765 15.385 30.8 50.0 66.7 100.0 200.0 400.0 25000 1.250 2.500 5.000 10.417 14.706 19.231 38.5 62.5 83.3 125.0 250.0 500.0 35000 1.750 3.500 7.000 14.583 20.588 26.923 53.8 87.5 116.7 175.0 350.0 700.0 50000 2.500 5.000 10.000 20.833 29.412 38.462 76.9 125.0 166.7 250.0 500.0 1000.0 75000 3.750 7.500 15.000 31.250 44.118 57.692 115.4 187.5 250.0 375.0 750.0 1500.0 100000 5.000 10.000 20.000 41.667 58.824 76.923 153.8 250.0 333.3 500.0 1000.0 2000.0 DAE below 0.2, or below 5.

The higher comparative ratios described herein further suggest that a given dose of RPPX can contain a certain amount of PPX impurity before exceeding the acceptable DAE. For example, Table 3 shows that a 25 mg dose of RPPX results in 0.00125 DAE at a comparative ratio of 20,000 as suggested by the NOAEL ratio of RPPX to PPX in the dog studies, assuming a 100% chiral purity of RPPX. Theoretically, an additional 1.4 mg of PPX could be added without exceeding the DAE for a single dose MTD of PPX, while an additional 0.045 mg of PPX could be added before exceeding the preferable NOAEL dose amount of PPX. These compositions would be 96% pure and 99.8% pure. By contrast, a 25 mg of 100% pure RPPX would result in 2.78 DAE, using the comparative binding affinity ratio of 9 from the literature, suggesting that even 100% purity would be insufficient to avoid adverse side effects. Hence, the present invention further provides particular doses of RPPX which unexpectedly tolerate small amounts of PPX impurities.

Further Definitions

It must also be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to a “salt” is a reference to one or more organic solvents and equivalents thereof known to those skilled in the art, and so forth.

As used herein, the term “about” means plus or minus 10% of the numerical value of the number with which it is being used. Therefore, about 50% means in the range of 45%-55%. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

As used herein, the term “comparative binding affinity ratio” refers to the binding affinity at the D₂ or D₃ dopamine receptors (IC₅₀ value) of RPPX divided by the binding affinity at the D₂ or D₃ dopamine receptors (IC₅₀ value) of PPX. In some embodiments, the comparative binding affinity ratio refers to the ratio of the IC₅₀ values at the D₂ receptor. In some embodiments, the comparative binding affinity ratio refers to the ratio of the IC₅₀ values at the D₃ receptor.

As used herein, the term “comparative ratio” refers one of the following: 1) the ratio of the IC₅₀ values at the D₂ or D₃ receptors for RPPX to PPX; 2); the ratio of MTD amounts for RPPX to PPX; or 3) the ratio of NOAEL dose amounts for RPPX to PPX.

As used herein, the term “controlled release” refers to a dosage form that release drug at a controlled rate over an extended period of time, preferably more than about 12 hours, more preferably about 24 hours.

As used herein, the term “daily dose amount” refers to the amount of drug per day that is administered or prescribed to a patient. This amount can be administered in multiple unit doses or in a single unit dose, in a single time during the day or at multiple times during the day. Preferably, the daily dose amount is administered in a modified release pharmaceutical composition.

As used herein, the term “dopaminergic activity equivalent” (DAE) refers to the measure of activity at the dopamine receptors which is equivalent to the activity of 1 mg of PPX at the dopamine receptors.

A “dose amount” as used herein, is generally equal to the dosage of the active ingredient which may be administered once per day, or may be administered several times a day (e.g. the unit dose is a fraction of the desired daily dose). For example, a non-effective dose amount of 0.5 mg/day of PPX may be administered as 1 dose of 0.5 mg, 2 doses of 0.25 mg each or 4 doses of 0.125 mg. The term “unit dose” as used herein may be taken to indicate a discrete amount of the therapeutic composition which comprises a predetermined amount of the active compound. The amount of the active ingredient is generally equal to the dosage of the active ingredient which may be administered once per day, or may be administered several times a day (e.g. the unit dose is a fraction of the desired daily dose). The unit dose may also be taken to indicate the total daily dose, which may be administered once per day or may be administered as a convenient fraction of such a dose (e.g. the unit dose is the total daily dose which may be given in fractional increments, such as, for example, one-half or one-third the dosage).

As used herein, the terms “enantiomers”, “stereoisomers” and “optical isomers” may be used interchangeably, and refer to molecules which contain an asymmetric or chiral center and are non-superimposable mirror images of one another. As used herein, the term “chirally pure” or “enantiomerically pure” may be taken to indicate that the compound contains at least 99.95% of a single optical isomer. The term “enantiomerically enriched”, unless a number is mentioned, may be taken to indicate that at least 51% of the material is a single enantiomer. The term “enantiomeric enrichment” as used herein refers to an increase in the amount of one enantiomer as compared to the other. A “racemic” mixture is a mixture of equal amounts of (R)- and (S)-enantiomers of a chiral molecule.

“Extended release” and/or “sustained release” as used herein refers to a dosage form that releases a drug (or drugs) over an extended period of time such that the frequency of administration may be reduced as compared to immediate release dosage forms, particularly a dosage form that allows at least a twofold reduction in dosing frequency of a drug as compared to that drug presented as a conventional dosage form (e.g. as a solution or prompt drug-releasing, conventional solid dosage form). For example, an extended release dosage form may release the drug over a period of time of about more than 12 hours, preferably about 24 hours. Extended release is used interchangeably herein with sustained release and slow release.

As used herein, a “kit” refers to one or more pharmaceutical compositions and instructions for administration or prescription of the one or more compositions. The instructions may consist of product insert, instructions on a package of one or more pharmaceutical compositions, or any other instruction.

As used herein, the term “Mirapex®” refers to tablets containing pramipexole dihydrochloride, which has the chemical name, (S)-2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole dihydrochloride monohydrate.

“Modified release” as used herein, refers to a dosage form for which the drug release characteristics of time course and/or location are chosen to accomplish therapeutic or convenience objectives not offered by conventional dosage forms such as solutions, ointments, or promptly dissolving dosage forms. Modified release of active ingredient(s) allows simplification of the patient's administration scheme by reducing the amount of recommended daily intakes, improves patient's compliance, and attenuates adverse events, e.g., related to high plasma peaks. Modified release pharmaceutical preparations regulate the release of the incorporated active ingredient or ingredients over time and comprise formulations with a controlled, a prolonged, a sustained, a slow or an extended release, so they accomplish therapeutic or convenience objectives not offered by conventional dosage forms such as solutions or promptly dissolving dosage forms. Preferably, the modified release formulations are capable of release a therapeutically effective amount of the drug, RPPX, over an extended period of time, preferably about more than twelve hours, more preferably about twenty-four hours. Controlled release, extended release, and sustained release dosage forms and their combinations are types of modified release dosage forms. Unless otherwise indicated all such terms are used interchangeably.

As used herein, the term “naïve patient” refers to a patient that has not previously received treatment (either RPPX or PPX) or who has not received a titration regimen previous to receiving a starting dose.

As used herein, the term “neuroprotectant” refers to any agent that may prevent or slow the progression of neuronal degeneration and/or may prevent neuronal cell death.

The term “patient” and “subject” are interchangeable and may be taken to mean any living organism which may be treated with compounds of the present invention. As such, the terms “patient” and “subject” may include, but is not limited to, any non-human mammal, primate or human. In some embodiments, the “patient” or “subject” is a mammal, such as mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, primates, or humans. In some embodiments, the patient or subject is an adult, child or infant. In some embodiments, the patient or subject is a human.

As used herein, the term “pharmaceutically acceptable salt” is meant to indicate those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. The pharmaceutically acceptable salts of the compounds can be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. Generally, such salts can be prepared by-reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, non-aqueous media like diethyl ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 20th ed., Lippincott Williams & Wilkins, Baltimore, Md., p. 704 (2000) and Berge et al. (1977) J. Pharm. Sciences, Vol 6. 1-19.

The term “pharmaceutical composition” shall mean a composition comprising at least one active ingredient, whereby the composition is amenable to investigation for a specified, efficacious outcome in a mammal (for example, without limitation, a human). Those of ordinary skill in the art will understand and appreciate the techniques appropriate for determining whether an active ingredient has a desired efficacious outcome based upon the needs of the artisan.

As used herein, the term “RPPX” refers the (R)-enantiomer of pramipexole, or its pharmaceutically acceptable salt thereof preferably the R(+) enantiomer of pramipexole, or pharmaceutically acceptable salt thereof. “RPPX” can also include the hydrate of the (R)-enantiomer of pramipexole, or pharmaceutically acceptable salt thereof. In some embodiments, RPPX is RPPX dihydrochloride monohydrate.

As used herein, the term “PPX” refers to pramipexole, which is an (S)-enantiomer, or pharmaceutically acceptable salt thereof, “PPX” can also include the hydrate of pramipexole, or pharmaceutically acceptable salt thereof.

As used herein, the term “salt” of the RPPX as used herein is any acid addition salt, preferably a pharmaceutically acceptable acid addition salt, including but not limited to, halogenic acid salts such as, for example, hydrobromic, hydrochloric, hydrofluoric and hydroiodic acid salt; an inorganic acid salt such as, for example, nitric, perchloric, sulfuric and phosphoric acid salt; an organic acid salt such as, for example, sulfonic acid salts (methanesulfonic, trifluoromethan sulfonic, ethanesulfonic, benzenesulfonic or p-toluenesulfonic), acetic, malic, fumaric, succinic, citric, benzoic, gluconic, lactic, mandelic, mucic, pamoic, pantothenic, oxalic and maleic acid salts; and an amino acid salt such as aspartic or glutamic acid salt. The acid addition salt may be a mono- or di-acid addition salt, such as a di-hydrohalogenic, di-sulfuric, di-phosphoric or di-organic acid salt. In all cases, the acid addition salt is used as an achiral reagent which is not selected on the basis of any expected or known preference for interaction with or precipitation of a specific optical isomer of the products of this invention (e.g. as opposed to the specific use of D(+) tartaric acid in the prior art, which may preferentially precipitate the RPPX).

As used herein, the term “starting daily dose amount” refers to the amount of pramipexole per day that is administered or prescribed to a patient beginning pramipexole treatment, who has not previously been subjected to a titration regimen of pramipexole. This amount can be administered in multiple unit doses or in a single unit dose, in a single time during the day or at multiple times during the day.

“Therapeutically effective amount” as used herein refers to the amount of active compound or pharmaceutical agent that elicits a biological or medicinal response in a tissue, system, animal, individual or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes one or more of the following: (1) preventing the disease; for example, preventing a disease, condition or disorder in an individual that may be predisposed to the disease, condition or disorder but does not yet experience or display the pathology or symptomatology of the disease, (2) inhibiting the disease; for example, inhibiting a disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., arresting or slowing further development of the pathology and/or symptomatology), and (3) ameliorating the disease; for example, ameliorating a disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., reversing or reducing the pathology and/or symptomatology).

The term “treating” may be taken to mean prophylaxis of a specific disorder, disease or condition, alleviation of the symptoms associated with a specific disorder, disease or condition and/or prevention of the symptoms associated with a specific disorder, disease or condition. In some embodiments, the term refers to slowing the progression of the disorder, disease or condition or alleviating the symptoms associated with the specific disorder, disease or condition. In some embodiments, the term refers to slowing the progression of the disorder, disease or condition. In some embodiments, the term refers to alleviating the symptoms associated with the specific disorder, disease or condition. In some embodiments, the term refers to restoring function which was impaired or lost due to a specific disorder, disease or condition.

The term “trituration” may be taken to indicate a method of solidifying a chemical compound. Trituration involves agitating the compound by stirring, beating or a method of the like until the chemical compound forms a crystalline solid or precipitate. This solid may act to seed the remaining chemical compound in solution, causing it to precipitate or crystallize from solution.

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred methods, devices, and materials are now described.

Modified Release Pharmaceutical Compositions

The high chiral purity of the R-(+) enantiomer of pramipexole used herein, or RPPX, allows for therapeutic compositions that may have a wide individual and daily dose range. As such, in a first aspect, the present invention provides a modified release pharmaceutical composition comprising RPPX. The composition may further comprise a pharmaceutically acceptable carrier.

The embodiments for amounts of RPPX in the modified release composition, chiral purity. NOAEL, NOEL, DAE, NAE and dosage form, which are described herein separately for the sake of brevity, can be joined in any suitable combination.

In some embodiments, the amount of RPPX may be from about 0.01 mg/kg/day to about 10,000 mg/kg/day, from about 1 mg/kg/day to about 1,000 mg/kg/day, from about 0.1 mg/kg/day to about 1,000 mg/kg/day, from about 1 mg/kg/day to about 1,000 mg/kg/day, from about 1,000 mg/kg/day to about 10,000 mg/kg/day, or from about 1 mg/kg/day to about 100 mg/kg/day. In some embodiments, the amount of RPPX may be from about 3 mg/kg/day to about 70 mg/kg/day. In some embodiments, amount of RPPX may be from about 7 mg/kg/day to about 40 mg/kg/day. In some embodiment, the amount of RPPX may be from about 3 mg/kg/day to about 50 mg/kg/day. In some embodiments, the dosage may be 10 mg/day to 1,500 mg/day, more preferably 100 mg/day to 600 mg/day. The amount of RPPX in the modified release compositions may preferably be about 50 mg to about 5,000 mg, from about 100 mg to about 3,000 mg, from about 300 mg to about 1,500 mg, from about 500 mg to about 1,000 mg. In some embodiments, the amount of RPPX in the modified release compositions may be about from about 25 mg to about 5,000 mg, from about 50 mg to about 5,000 mg, from about 100 mg to about 5,000 mg, from about 200 mg to about 5,000 mg, from about 250 mg to about 5,000 mg, from about 300 mg to about 5,000 mg, from about 400 mg to about 5,000 mg, from 450 mg to about 5,000 mg, from about 200 mg, to about 3,000 mg, from about 250 mg to about 3,000 mg, from about 300 mg to about 3,000 mg, from about 400 mg to about 3,000 mg, from 450 mg to about 3,000 mg, from about 100 mg to about 1,000 mg, from about 200 mg to about 1,000 mg, from about 250 mg to about 1,000 mg, from about 300 mg to about 1,000 mg, from about 400 mg to about 1,000 mg, from about 600 mg to about 1,000 mg, or from 450 mg to about 1,000 mg. In some embodiments, the amount of RPPX is from about 600 mg to about 900 mg. Preferably, these doses may be administered in a modified release pharmaceutical composition that provides release of a dose of RPPX over preferably more than about twelve hours, and more preferably about twenty-four hours. In some embodiments, the amount of RPPX is from about 50 mg to about 5000 mg. In some embodiments, the amount of RPPX is from about 100 mg to about 3000 mg. In some embodiments, the amount of RPPX is from about 300 mg to about 1500 mg. In some embodiments, the amount of RPPX is from about 500 mg to about 1000 mg. In some embodiments, the modified release composition is suitable for oral administration. In some embodiments, the modified release composition is a solid oral dosage form.

The modified release composition may have a chiral purity for RPPX of at least 99.5%, preferably at least 99.6%, preferably at least 99.7%, preferably at least 99.8%, preferably at least 99.9%, preferably at least 99.95%, or more preferably at least 99.99%. In some embodiments, the chiral purity for RPPX is 100%. In some embodiments, the modified release composition has a chiral purity for RPPX of 99.9% or greater. In some embodiments, the modified release composition has a chiral purity for RPPX of 99.95% or greater. In some embodiments, the modified release composition has a chiral purity for RPPX of 99.99% or greater.

In some embodiments, the modified release composition is suitable for oral administration. In some embodiments, the modified release composition is a solid oral dosage form. In some embodiments, the modified release composition is a capsule. In some embodiments, the modified release composition is a tablet.

In another aspect, the present invention relates to modified release compositions which are chirally pure for RPPX. In some embodiments, the amount of RPPX may be from about 0.01 mg/kg/day to about 10,000 mg/kg/day, from about 1 mg/kg/day to about 1,000 mg/kg/day, from about 0.1 mg/kg/day to about 1,000 mg/kg/day, from about 1 mg/kg/day to about 1,000 mg/kg/day, from about 1,000 mg/kg/day to about 10,000 mg/kg/day, or from about 1 mg/kg/day to about 100 mg/kg/day. In some embodiments, the amount of RPPX may be from about 3 mg/kg/day to about 70 mg/kg/day. In some embodiments, the amount of RPPX may be from about 7 mg/kg/day to about 40 mg/kg/day. In some embodiment, the amount of RPPX may be from about 3 mg/kg/day to about 50 mg/kg/day. In some embodiments, the dosage may be 10 mg/day to 1,500 mg/day, more preferably 100 mg/day to 600 mg/day. In some embodiments, the compositions are administered in doses of from about 50 mg to about 5,000 mg, from about 100 mg to about 3,000 mg, from about 300 mg to about 1,500 mg, or from about 500 mg to about 1,000 mg of RPPX. In some embodiments, the modified release compositions are administered in doses of from 50 mg to about 5,000 mg, from about 100 mg to about 5,000 mg, from about 200 mg to about 5,000 mg, from about 250 mg to about 5,000 mg, from about 300 mg to about 5,000 mg, from about 400 mg to about 5,000 mg, from 450 mg to about 5,000 mg, from about 200 mg, to about 3,000 mg, from about 250 mg to about 3,000 mg, from about 300 mg to about 3,000 mg, from about 400 mg to about 3,000 mg, from 450 mg to about 3,000 mg, from about 100 mg to about 1,000 mg, from about 200 mg to about 1,000 mg, from about 250 mg to about 1,000 mg, from about 300 mg to about 1,000 mg, from about 400 mg to about 1,000 mg, from about 600 mg to about 1,000 mg, or from 450 mg to about 1,000 mg of RPPX. In some embodiments, the amount of RPPX is from about 600 mg to about 900 mg. Preferably, these doses may be administered in a modified release pharmaceutical composition that provides release of the dose of RPPX over preferably more than twelve hours, and more preferably at least about twenty-four hours. These doses preferably are in preparations which have a chemical purity of 97% or greater and a chiral purity for RPPX of 99.6% or greater, 99.7% or greater, 99.8% or greater, 99.9% or greater, preferably 99.95% or greater and more preferably 99.99% or greater. In a preferred embodiment, the modified release compositions may have a chiral purity for RPPX of 100%. The modified release compositions may further comprise a carrier. The modified release compositions of the present invention may be administered orally, preferably as a solid oral dose, and more preferably as a solid oral dose that may be a capsule or tablet. In preferred embodiments, the modified release compositions of the present invention may be formulated as tablets for oral administration.

In another aspect, the present invention further provides a modified release composition comprising a therapeutically effective amount of RPPX. The composition may further comprise a pharmaceutically acceptable carrier.

In some embodiments, the therapeutically effective amount of RPPX may be from about 0.01 mg/kg/day to about 10,000 mg/kg/day, from about 1 mg/kg/day to about 1,000 mg/kg/day, from about 0.1 mg/kg/day to about 1,000 mg/kg/day, from about 1 mg/kg/day to about 1,000 mg/kg/day, from about 1,000 mg/kg/day to about 10,000 mg/kg/day, or from about 1 mg/kg/day to about 100 mg/kg/day. In some embodiments, the therapeutically effective amount of RPPX may be from about 3 mg/kg/day to about 70 mg/kg/day. In some embodiments, the therapeutically effective amount of RPPX may be from about 7 mg/kg/day to about 40 mg/kg/day. In some embodiment, the therapeutically effective amount of RPPX may be from about 3 mg/kg/day to about 50 mg/kg/day. In some embodiments, the dosage may be 10 mg/day to 1,500 mg/day, more preferably 100 mg/day to 600 mg/day. The therapeutically effective amount of RPPX in the modified release compositions may preferably be about 50 mg to about 5,000 mg, from about 100 mg to about 3,000 mg, from about 300 mg to about 1,500 mg, from about 500 mg to about 1,000 mg. In some embodiments, the therapeutically effective amount of RPPX in the modified release compositions may be about from about 50 mg to about 5,000 mg, from about 100 mg to about 5,000 mg, from about 200 mg to about 5,000 mg, from about 250 mg to about 5,000 mg, from about 300 mg to about 5,000 mg, from about 400 mg to about 5,000 mg, from 450 mg to about 5,000 mg, from about 200 mg, to about 3,000 mg, from about 250 mg to about 3,000 mg, from about 300 mg to about 3,000 mg, from about 400 mg to about 3,000 mg, from 450 mg to about 3,000 mg, from about 100 mg to about 1,000 mg, from about 200 mg to about 1,000 mg, from about 250 mg to about 1,000 mg, from about 300 mg to about 1,000 mg, from about 400 mg to about 1,000 mg, from about 600 mg to about 1,000 mg, or from 450 mg to about 1,000 mg. In some embodiments, the amount of RPPX is from about 600 mg to about 900 mg. Preferably, these doses may be administered in a modified release pharmaceutical composition that provides release of a dose of RPPX over preferably more than about twelve hours, and more preferably about twenty-four hours. In some embodiments, the therapeutically effective amount of RPPX is from about 50 mg to about 5000 mg. In some embodiments, the therapeutically effective amount of RPPX is from about 100 mg to about 3000 mg. In some embodiments, the therapeutically effective amount of RPPX is from about 300 mg to about 1500 mg. In some embodiments, the therapeutically effective amount of RPPX is from about 500 mg to about 1000 mg. In some embodiments, the modified release composition is suitable for oral administration. In some embodiments, the modified release composition is a solid oral dosage form.

The modified release composition may have a chiral purity for RPPX of at least 99.5%, preferably at least 99.6%, preferably at least 99.7%, preferably at least 99.8%, preferably at least 99.9%, preferably at least 99.95%, or more preferably at least 99.99%. In some embodiments, the chiral purity for RPPX is 100%. In some embodiments, the modified release composition has a chiral purity for RPPX of 99.9% or greater. In some embodiments, the modified release composition has a chiral purity for RPPX of 99.95% or greater. In some embodiments, the modified release composition has a chiral purity for RPPX of 99.99% or greater.

In some embodiments, the modified release composition is suitable for oral administration. In some embodiments, the modified release composition is a solid oral dosage form. In some embodiments, the modified release composition is a capsule. In some embodiments, the modified release composition is a tablet.

In an additional aspect, the present invention provides a modified release composition consisting essentially of a therapeutically effective amount of RPPX, wherein the chiral purity for the RPPX is 99.9%, or greater. In some embodiments, the chiral purity for RPPX is 99.95% or greater. In some embodiments, the chiral purity for RPPX is 99.99% or greater. In some embodiments, the chiral purity for RPPX is 100%.

In some embodiments, the modified release composition is suitable for oral administration. In some embodiments, the modified release composition is a solid oral dosage form. In some embodiments, the modified release composition is a capsule. In some embodiments, the modified release composition is a tablet.

In a further aspect, the present invention further provides a modified release composition comprising a therapeutically effective amount of RPPX and a non-effective dose amount of PPX. The composition may further comprise a pharmaceutically acceptable carrier.

In some embodiments, the amount of RPPX may be from about 0.01 mg/kg/day to about 10,000 mg/kg/day, from about 1 mg/kg/day to about 1,000 mg/kg/day, from about 0.1 mg/kg/day to about 1,000 mg/kg/day, from about 1 mg/kg/day to about 1,000 mg/kg/day, from about 1,000 mg/kg/day to about 10,000 mg/kg/day, or from about 1 mg/kg/day to about 100 mg/kg/day. In some embodiments, the amount of RPPX may be from about 3 mg/kg/day to about 70 mg/kg/day. In some embodiments, the amount of RPPX may be from about 7 mg/kg/day to about 40 mg/kg/day. In some embodiment, the amount of RPPX may be from about 3 mg/kg/day to about 50 mg/kg/day. In some embodiments, the dosage may be 10 mg/day to 1,500 mg/day, more preferably 100 mg/day to 600 mg/day. The amount of RPPX in the modified release compositions may preferably be about 50 mg to about 5,000 mg, from about 100 mg to about 3,000 mg, from about 300 mg to about 1,500 mg, from about 500 mg to about 1,000 mg. In some embodiments, the amount of RPPX in the modified release compositions may be about from about 25 mg to about 5,000 mg, from about 50 mg to about 5,000 mg, from about 100 mg to about 5,000 mg, from about 200 mg to about 5,000 mg, from about 250 mg to about 5,000 mg, from about 300 mg to about 5,000 mg, from about 400 mg to about 5,000 mg, from 450 mg to about 5,000 mg, from about 200 mg, to about 3,000 mg, from about 250 mg to about 3,000 mg, from about 300 mg to about 3,000 mg, from about 400 mg to about 3,000 mg, from 450 mg to about 3,000 mg, from about 100 mg to about 1,000 mg, from about 200 mg to about 1,000 mg, from about 250 mg to about 1,000 mg, from about 300 mg to about 1,000 mg, from about 400 mg to about 1,000 mg, from about 600 mg to about 1,000 mg, or from 450 mg to about 1,000 mg. Preferably, these doses may be administered in a modified release pharmaceutical composition that provides release of a dose of RPPX over preferably more than about twelve hours, and more preferably about twenty-four hours. In some embodiments, the amount of RPPX is from about 50 mg to about 5000 mg. In some embodiments, the amount of RPPX is from about 100 mg to about 3000 mg. In some embodiments, the amount of RPPX is from about 300 mg to about 1500 mg. In some embodiments, the amount of RPPX is from about 500 mg to about 1000 mg. In some embodiments, the amount of RPPX is from about 600 mg to about 900 mg. In some embodiments, the modified release composition is suitable for oral administration. In some embodiments, the composition is a solid oral dosage form.

The modified release composition may have a chiral purity for RPPX of at least 99.5%, preferably at least 99.6%, preferably at least 99.7%, preferably at least 99.8%, preferably at least 99.9%, preferably at least 99.95%, or more preferably at least 99.99%. In some embodiments, the chiral purity for RPPX is 100%. In some embodiments, the modified release composition has a chiral purity for RPPX of 99.9% or greater. In some embodiments, the modified release composition has a chiral purity for RPPX of 99.95% or greater. In some embodiments, the modified release composition has a chiral purity for RPPX of 99.99% or greater.

In embodiments, the non-effective dose amount of PPX is an amount that does not exceed about 1.0 mg. In more preferred embodiments, the non-effective dose amount of PPX is an amount that does not exceed about 0.75 mg, about 0.5 mg, about 0.25 mg, or about 0.125 mg. In some embodiments, the non-effective dose amount of PPX is less than about 0.125 mg.

In some embodiments, the modified release composition is suitable for oral administration. In some embodiments, the modified release composition is a solid oral dosage form. In some embodiments, the modified release composition is a capsule. In some embodiments, the modified release composition is a tablet.

In another aspect, the present invention provides a modified release pharmaceutical composition comprising a therapeutically effective amount of RPPX and a non-effective dose amount of PPX administered in a unit dose form. Preferable unit dose forms include those suitable for oral administration, including but not limited to, capsules, tablets and the like. Table 5 shows various exemplary embodiments. Shown in each column of Table 5 is the amount of PPX that may be co-administered in a non-effective dose amount as a function of the chiral purity of the composition for the (R)-enantiomer of pramipexole. The therapeutically effective amount of RPPX may preferably be about 50 mg to about 5,000 mg, from about 100 mg to about 3,000 mg, from about 300 mg to about 1,500 mg, from about 500 mg to about 1,000 mg. In some embodiments, therapeutically effective amount of RPPX may be about from about 50 mg to about 5,000 mg, from about 100 mg to about 5,000 mg, from about 200 mg to about 5,000 mg, from about 250 mg to about 5,000 mg, from about 300 mg to about 5,000 mg, from about 400 mg to about 5,000 mg, from 450 mg to about 5,000 mg, from about 200 mg, to about 3,000 mg, from about 250 mg to about 3,000 mg, from about 300 mg to about 3,000 mg, from about 400 mg to about 3,000 mg, from 450 mg to about 3,000 mg, from about 100 mg to about 1,000 mg, from about 200 mg to about 1,000 mg, from about 250 mg to about 1,000 mg, from about 300 mg to about 1,000 mg, from about 400 mg to about 1,000 mg from about 600 mg to about 1,000 mg, or from 450 mg to about 1,000 mg. In some embodiments, the therapeutically effective amount of RPPX is from about 600 mg to about 900 mg.

The non-effective dose amount of PPX may be preferably below 1.0 mg/day, below 0.5 mg/day, and below 0.125 mg/day. Thus, as a non-limiting example, a dose of 500 mg/day administered to a patient as a single unit dose may have a chiral purity for the R(+) enantiomer of pramipexole of at least about 99.80% so that the non-effective dose amount of PPX may remain below 1.0 mg/day, more preferably about 99.90% so that the non-effective dose amount of PPX may remain below 0.5 mg/day, and more preferably about 99.975% so that the non-effective dose amount of PPX may remain below 0.125 mg/day. The embodiments for the therapeutically effective amount of RPPX, the non-effective dose amount of PPX, and the chiral purity embodiments listed herein may be combined in any suitable combination. With reference to Table 5, any combination of chiral purity and unit dose may be used which allows for the desired combination of a therapeutically effective amount of RPPX and a non-effective dose amount of PPX as stated herein.

In some embodiments, the modified release pharmaceutical composition is suitable for oral administration and comprises an amount of RPPX greater than 100 mg and a non-effective dose amount of PPX that is less than about 0.125 mg. Another preferred embodiment is a modified release pharmaceutical composition suitable for oral administration comprising an amount of RPPX greater than 250 mg and a non-effective dose amount of PPX that is less than about 0.125 mg. Yet another preferred embodiment of the invention is a modified release pharmaceutical composition suitable for oral administration comprising an amount of RPPX greater than 500 mg and a non-effective dose amount of PPX that is less than about 0.125 mg. Preferred modified release pharmaceutical compositions for oral administration include tablets, capsules and the like.

In some embodiments, the modified release pharmaceutical composition is formulated as a tablet suitable for oral administration and comprises an amount of RPPX greater than 50 mg and a non-effective dose amount of PPX that is less than about 0.50 mg, preferably an amount of RPPX greater than 100 mg and a non-effective dose amount of PPX that is less than about 0.50 mg, and more preferably an amount of RPPX greater than 250 mg and a non-effective dose amount of PPX that is less than about 0.50 mg. Another preferred embodiment is a modified release pharmaceutical composition formulated as a tablet suitable for oral administration comprising an amount of RPPX greater than 500 mg and a non-effective dose amount of PPX that is less than about 0.50 mg.

TABLE 5 Preferred non-effective dose amounts of PPX based on the chiral purity of the composition for RPPX

A preferred non-effective dose amount of the PPX may be below 1.0 mg; more preferably below 0.5 mg, and more preferably below 0.125 mg.

Another embodiment of the invention is a modified release pharmaceutical composition formulated as a tablet suitable for oral administration comprising an amount of RPPX greater than 50 mg and a non-effective dose amount of PPX that is less than about 0.25 mg, preferably an amount of RPPX greater than 100 mg and a non-effective dose amount of PPX that is less than about 0.25 mg, and more preferably an amount of RPPX greater than 250 mg and a non-effective dose amount of PPX that is less than about 0.25 mg. Another preferred embodiment is a modified release pharmaceutical composition formulated as a tablet suitable for oral administration comprising an amount of RPPX greater than 500 mg and a non-effective dose amount of PPX that is less than about 0.25 mg.

In another aspect, the present invention provides a modified release composition comprising a therapeutically effective amount of RPPX and a no observable adverse effect level (NOAEL) dose amount of PPX. The therapeutic composition may further comprise a pharmaceutically acceptable carrier.

In some embodiments, the amount of RPPX may be from about 0.01 mg/kg/day to about 10,000 mg/kg/day, from about 1 mg/kg/day to about 1,000 mg/kg/day, from about 0.1 mg/kg/day to about 1,000 mg/kg/day, from about 1 mg/kg/day to about 1,000 mg/kg/day, from about 1,000 mg/kg/day to about 10,000 mg/kg/day, or from about 1 mg/kg/day to about 100 mg/kg/day. In some embodiments, the amount of RPPX may be from about 3 mg/kg/day to about 70 mg/kg/day. In some embodiments, the amount of RPPX may be from about 7 mg/kg/day to about 40 mg/kg/day. In some embodiment, the amount of RPPX may be from about 3 mg/kg/day to about 50 mg/kg/day. In some embodiments, the dosage may be 10 mg/day to 1,500 mg/day, more preferably 100 mg/day to 600 mg/day. The amount of RPPX in the modified release compositions may preferably be about 50 mg to about 5,000 mg, from about 100 mg to about 3,000 mg, from about 300 mg to about 1,500 mg, from about 500 mg to about 1,000 mg. In some embodiments, the amount of RPPX in the modified release compositions may be about from about 25 mg to about 5,000 mg, from about 50 mg to about 5,000 mg, from about 100 mg to about 5,000 mg, from about 200 mg to about 5,000 mg, from about 250 mg to about 5,000 mg, from about 300 mg to about 5,000 mg, from about 400 mg to about 5,000 mg, from 450 mg to about 5,000 mg, from about 200 mg, to about 3,000 mg, from about 250 mg to about 3,000 mg, from about 300 mg to about 3,000 mg, from about 400 mg to about 3,000 mg, from 450 mg to about 3,000 mg, from about 100 mg to about 1,000 mg, from about 200 mg to about 1,000 mg, from about 250 mg to about 1,000 mg, from about 300 mg to about 1,000 mg, from about 400 mg to about 1,000 mg, from about 600 mg to about 1,000 mg, or from 450 mg to about 1,000 mg. In some embodiments, the amount of RPPX is from about 600 mg to about 900 mg. In some embodiments, the amount of RPPX is from about 50 mg to about 5000 mg. In some embodiments, the amount of RPPX is from about 100 mg to about 3000 mg. In some embodiments, the amount of RPPX is from about 300 mg to about 1500 mg. In some embodiments, the amount of RPPX is from about 500 mg to about 1000 mg. In some embodiments, the composition is suitable for oral administration. In some embodiments, the modified release composition is a solid oral dosage form.

The modified release composition may have a chiral purity for RPPX of at least 99.5%, preferably at least 99.6%, preferably at least 99.7%, preferably at least 99.8%, preferably at least 99.9%, preferably at least 99.95%, or more preferably at least 99.99%. In some embodiments, the chiral purity for RPPX is 100%. In some embodiments, the modified release composition has a chiral purity for RPPX of 99.9% or greater. In some embodiments, the modified release composition has a chiral purity for RPPX of 99.95% or greater. In some embodiments, the modified release composition has a chiral purity for RPPX of 99.99% or greater.

In some embodiments, the no observable adverse effect level dose amount of PPX is less than about 4.50 mg. In some embodiments, the no observable adverse effect level amount of PPX is less than about 0.15 mg. In some embodiments, the no observable adverse effect level amount of PPX is less than about 0.15 mg.

In some embodiments, the modified release composition is suitable for oral administration. In some embodiments, the modified release composition is a solid oral dosage form. In some embodiments, the modified release composition is a capsule. In some embodiments, the modified release composition is a tablet.

In an additional aspect, the present invention provides a modified release pharmaceutical composition comprising a therapeutically effective amount of RPPX and a NOAEL dose amount of PPX administered in a unit dose form. Preferable unit dose forms include those suitable for oral administration, including but not limited to, capsules, tablets and the like. Table 6 shows various exemplary embodiments. Shown in each column of Table 6 is the amount of PPX that may be co-administered in a NOAEL dose amount as a function of the chiral purity of the modified release composition for the R(+) enantiomer of pramipexole. The therapeutically effective amount of RPPX may preferably be about 50 mg to about 5,000 mg, preferably from about 100 mg to about 3,000 mg, preferably from about 300 mg to about 1,500 mg, more preferably from about 500 mg to about 1,000 mg. In some embodiments, therapeutically effective amount of RPPX may be about from about 50 mg to about 5,000 mg, from about 100 mg to about 5,000 mg, from about 200 mg to about 5,000 mg, from about 250 mg to about 5,000 mg, from about 300 mg to about 5,000 mg, from about 400 mg to about 5,000 mg, from 450 mg to about 5,000 mg, from about 200 mg to about 3,000 mg, from about 250 mg to about 3,000 mg, from about 300 mg to about 3,000 mg, from about 400 mg to about 3,000 mg, from 450 mg to about 3,000 mg, from about 100 mg to about 1,000 mg, from about 200 mg to about 1,000 mg, from about 250 mg to about 1,000 mg, from about 300 mg to about 1,000 mg, from about 400 mg to about 1,000 mg, from about 600 mg to about 1,000 mg, or from 450 mg to about 1,000 mg. In some embodiments, the amount of RPPX is from about 600 mg to about 900 mg.

The NOAEL dose of PPX may be preferably below 4.5 mg, preferably below 1.5 mg, or more preferably below 0.15 mg. Thus, as a non-limiting example, an embodiment of the invention may be a dose of 1,500 mg/day administered to a patient as a single unit dose which may have a chiral purity for the R(+) enantiomer of pramipexole that is at least about 99.967% so that the non-adverse dose of PPX may remain below 0.50 mg/dose. Alternatively, a dose of 1,500 mg/day administered to a patient as three individual doses of 500 mg may have a chiral purity of the RPPX that is at least about 99.90% so that the non-adverse dose of PPX may remain below 0.50 mg/dose or 1.5 mg/day. The embodiments for the therapeutically effective amount of RPPX, the NOAEL dose amount of PPX, and the chiral purity embodiments listed herein may be combined in any suitable combination. With reference to Table 6, any combination of chiral purity and unit dose may be used which allows for the desired combination of a therapeutically effective amount of RPPX and a non-adverse effect dose amount of PPX as stated herein.

In some embodiments, the modified release pharmaceutical composition is formulated as a tablet suitable for oral administration and comprises an amount of RPPX greater than 50 mg and a NOAEL dose amount of PPX that is less than about 0.15 mg, preferably an amount of RPPX greater than 100 mg and a NOAEL dose amount of PPX that is less than about 0.15 mg, and more preferably an amount of RPPX greater than 250 mg and a NOAEL dose amount of PPX that is less than about 0.15 mg. In some embodiments, the modified release pharmaceutical composition is formulated as a tablet suitable for oral administration and comprises an amount of RPPX greater than 500 mg and a NOAEL dose amount of PPX that is less than about 0.15 mg.

TABLE 6 Preferred no observable adverse effect level doses of PPX based on the chiral purity of the modified release composition for RPPX

A preferred no observable adverse effect level (NOAEL) dose amount of the PPX may be below 1.5 mg, preferably below 0.15 mg.

In some embodiments, the present invention provides a modified release composition for use as a neuroprotectant comprising a therapeutically effective amount of RPPX and a therapeutically effective amount of PPX. The modified release composition may further comprise a pharmaceutically acceptable carrier. The modified release composition may be useful in the treatment of diseases which may be alleviated by the action of a neuroprotectant. An additional embodiment of the invention is a therapeutic composition for use as a neuroprotectant comprising a therapeutically effective amount of RPPX and a therapeutically effective amount of PPX. The modified release composition may further comprise a pharmaceutically acceptable carrier. The therapeutic composition may be useful in the treatment of diseases related to neuronal degeneration or neuronal cell death.

In one embodiment, the modified release compositions of RPPX may be used to restore or improve neuronal, retinal and muscle function in adults and children. Further, the compositions of RPPX may be used to treat neurodegenerative diseases, or other diseases associated with mitochondrial dysfunction or increased oxidative stress. In some embodiments, the compositions of RPPX may treat neurodegenerative dementias, neurodegenerative movement disorders and ataxias, seizure disorders, motor neuron disorders or diseases, and inflammatory demyelinating disorders in adults and children. The compositions of the present invention may also be useful in the treatment of other disorders not listed herein, and any listing provided in this invention is for exemplary purposes only and is non-limiting.

In some embodiments, the modified release compositions which comprise RPPX may be effective as inhibitors of oxidative stress, inhibitors of lipid peroxidation, in the detoxification of oxygen radicals, and the normalization of mitochondrial function. Oxidative stress may be caused by an increase in oxygen and other free radicals, and has been associated with the fatal neurodegenerative disorder amyotrophic lateral sclerosis (ALS). ALS is a progressive neurodegenerative disorder involving the motor neurons of the cortex, brain stem, and spinal cord. About 10% of all ALS patients are familial cases, of which 20% have mutations in the superoxide dismutase 1 (SOD-1) gene. The SOD-1 enzyme may play a pivotal role in the pathogenesis and progression of familial amyotrophic lateral sclerosis (FALS). Recent studies also link the premature neuronal death associated with ALS to mutated mitochondrial genes which lead to abnormalities in functioning of the energy production pathways in mitochondria.

Modified release compositions which comprise RPPX may also be effective in the treatment of age related macular degeneration. As such, an embodiment of the invention may be a modified release composition comprising RPPX suitable for systemic administration, ocular administration or topical administration to the eye.

Thus, the neuroprotective effect of the modified release compositions of the present invention may derive at least in part from the ability of the (R)-enantiomer of pramipexole to prevent neural cell death by at least one of three mechanisms. First, the (R)-enantiomer of pramipexole may be capable of reducing the formation of reactive oxygen species in cells with impaired mitochondrial energy production. Second, the (R)-enantiomer of pramipexole may partially restore the reduced mitochondrial membrane potential that has been correlated with Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. Third, the (R)-enantiomer of pramipexole may block the cell death pathways which are produced by pharmacological models of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis diseases and mitochondrial impairment.

The modified release compositions of these several embodiments which comprise RPPX as an active agent may be effective as inhibitors of oxidative stress, inhibitors of lipid peroxidation, in the detoxification of oxygen radicals, and the normalization of mitochondrial function. Further, they may be effective as treatment for impaired motor function, and in degenerative diseases that may affect cardiac and striated muscle and retinal tissues. As such, they may be effective in the treatment of neurodegenerative diseases such as ALS, Parkinson's disease and Alzheimer's disease, and macular degeneration.

Another embodiment of the invention is a modified release composition consisting essentially of a therapeutically effective amount of RPPX and a non-effective dose amount of PPX. Another embodiment of the invention is a modified release composition consisting essentially of a therapeutically effective amount of RPPX and a NOAEL dose amount of PPX. Another embodiment of the invention is a modified release composition consisting of a therapeutically effective amount of RPPX and a non-effective dose amount of PPX. Such compositions may preferably be therapeutic or pharmaceutical compositions. Another embodiment of the invention is a modified release composition consisting of a therapeutically effective amount of RPPX and a NOAEL, dose amount of PPX. Such compositions may preferably be therapeutic or pharmaceutical compositions.

In another aspect, the present invention provides a modified release tablet comprising at least about 100 mg of RPPX and no more than about 4.5 mg of PPX. In some embodiments, the tablet comprises about 150 mg of RPPX. In some embodiments, the tablet comprises about 200 mg of RPPX. In some embodiments, the tablet comprises about 250 mg of RPPX. In some embodiments, the tablet comprises about 500 mg of RPPX. In some embodiments, the tablet comprises about 1000 mg of RPPX. In some embodiments, the tablet comprises no more than 3.0 mg of PPX. In some embodiments, the tablet comprises no more than 0.1 mg of PPX. In some embodiments, the tablet comprises no more than 0.9 mg of PPX. In some embodiments, the tablet comprises no more than 0.6 mg of PPX. In some embodiments, the tablet comprises no more than 0.375 mg of PPX. In some embodiments, the tablet further comprises a pharmaceutically acceptable carrier.

In some embodiments, the modified release tablet comprises about 150 mg of RPPX and no more than about 3.0 mg of PPX. In some embodiments, the tablet comprises about 150 mg of RPPX and no more than about 0.1 mg of PPX. In some embodiments, the tablet comprises about 150 mg of RPPX and no more than about 0.9 mg of PPX. In some embodiments, the tablet comprises about 150 mg of RPPX and no more than about 0.6 mg of PPX. In some embodiments, the tablet comprises about 150 mg of RPPX and no more than about 0.375 mg of PPX.

In some embodiments, the modified release tablet comprises about 200 mg of RPPX and no more than about 3.0 mg of PPX. In some embodiments, the tablet comprises about 200 mg of RPPX and no more than about 0.1 mg of PPX. In some embodiments, the tablet comprises about 200 mg of RPPX and no more than about 0.9 mg of PPX. In some embodiments, the tablet comprises about 200 mg of RPPX and no more than about 0.6 mg of PPX. In some embodiments, the tablet comprises about 200 mg of RPPX and no more than about 0.375 mg of PPX.

In some embodiments, the modified release tablet comprises about 250 mg of RPPX and no more than about 3.0 mg of PPX. In some embodiments, the tablet comprises about 250 mg of RPPX and no more than about 0.1 mg of PPX. In some embodiments, the tablet comprises about 250 mg of RPPX and no more than about 0.9 mg of PPX. In some embodiments, the tablet comprises about 250 mg of RPPX and no more than about 0.6 mg of PPX. In some embodiments, the tablet comprises about 250 mg of RPPX and no more than about 0.375 mg of PPX.

In some embodiments, the modified release tablet comprises about 500 mg of RPPX and no more than about 3.0 mg of PPX. In some embodiments, the tablet comprises about 500 mg of RPPX and no more than about 0.1 mg of PPX. In some embodiments, the tablet comprises about 500 mg of RPPX and no more than about 0.9 mg of PPX. In some embodiments, the tablet comprises about 500 mg of RPPX and no more than about 0.6 mg of PPX. In some embodiments, the tablet comprises about 500 mg of RPPX and no more than about 0.375 mg of PPX.

In some embodiments, the modified release tablet comprises about 1000 mg of RPPX and no more than about 3.0 mg of PPX. In some embodiments, the tablet comprises about 1000 mg of RPPX and no more than about 0.1 mg of PPX. In some embodiments, the tablet comprises about 1000 mg of RPPX and no more than about 0.9 mg of PPX. In some embodiments, the tablet comprises about 1000 mg of RPPX and no more than about 0.6 mg of PPX. In some embodiments, the tablet comprises about 1000 mg of RPPX and no more than about 0.375 mg of PPX.

The modified release tablet may have a chiral purity for RPPX of at least 99.5%, preferably at least 99.6%, preferably at least 99.7%, preferably at least 99.8%, preferably at least 99.9%, preferably at least 99.95%, or more preferably at least 99.99%. In some embodiments, the chiral purity for RPPX is 100%. In some embodiments, the tablet has a chiral purity for RPPX of 99.9% or greater. In some embodiments, the tablet has a chiral purity for RPPX of 99.95% or greater. In some embodiments, the tablet has a chiral purity for RPPX of 99.99% or greater.

In another aspect, the present invention provides a modified release capsule comprising at least about 100 mg of RPPX and no more than about 4.5 mg of PPX. In some embodiments, the capsule comprises about 150 mg of RPPX. In some embodiments, the capsule comprises about 200 mg of RPPX. In some embodiments, the capsule comprises about 250 mg of RPPX. In some embodiments, the capsule comprises about 500 mg of RPPX. In some embodiments, the capsule comprises about 1000 mg of RPPX. In some embodiments, the capsule comprises no more than 3.0 mg of PPX. In some embodiments, the capsule comprises no more than 0.1 mg of PPX. In some embodiments, the capsule comprises no more than 0.9 mg of PPX. In some embodiments, the capsule comprises no more than 0.6 mg of PPX. In some embodiments, the capsule comprises no more than 0.375 mg of PPX. In some embodiments, the capsule further comprises a pharmaceutically acceptable carrier.

In some embodiments, the modified release capsule comprises about 150 mg of RPPX and no more than about 3.0 mg of PPX. In some embodiments, the capsule comprises about 150 mg of RPPX and no more than about 0.1 mg of PPX. In some embodiments, the capsule comprises about 150 mg of RPPX and no more than about 0.9 mg of PPX. In some embodiments, the capsule comprises about 150 mg of RPPX and no more than about 0.6 mg of PPX. In some embodiments, the capsule comprises about 150 mg of RPPX and no more than about 0.375 mg of PPX.

In some embodiments, the modified release capsule comprises about 200 mg of RPPX and no more than about 3.0 mg of PPX. In some embodiments, the capsule comprises about 200 mg of RPPX and no more than about 0.1 mg of PPX. In some embodiments, the capsule comprises about 200 mg of RPPX and no more than about 0.9 mg of PPX. In some embodiments, the capsule comprises about 200 mg of RPPX and no more than about 0.6 mg of PPX. In some embodiments, the capsule comprises about 200 mg of RPPX and no more than about 0.375 mg of PPX.

In some embodiments, the modified release capsule comprises about 250 mg of RPPX and no more than about 3.0 mg of PPX. In some embodiments, the capsule comprises about 250 mg of RPPX and no more than about 0.1 mg of PPX. In some embodiments, the capsule comprises about 250 mg of RPPX and no more than about 0.9 mg of PPX. In some embodiments, the capsule comprises about 250 mg of RPPX and no more than about 0.6 mg of PPX. In some embodiments, the capsule comprises about 250 mg of RPPX and no more than about 0.375 mg of PPX.

In some embodiments, the modified release capsule comprises about 500 mg of RPPX and no more than about 3.0 mg of PPX. In some embodiments, the capsule comprises about 500 mg of RPPX and no more than about 0.1 mg of PPX. In some embodiments, the capsule comprises about 500 mg of RPPX and no more than about 0.9 mg of PPX. In some embodiments, the capsule comprises about 500 mg of RPPX and no more than about 0.6 mg of PPX. In some embodiments, the capsule comprises about 500 mg of RPPX and no more than about 0.375 mg of PPX.

In some embodiments, the modified release capsule comprises about 1000 mg of RPPX and no more than about 3.0 mg of PPX. In some embodiments, the capsule comprises about 1000 mg of RPPX and no more than about 0.1 mg of PPX. In some embodiments, the capsule comprises about 1000 mg of RPPX and no more than about 0.9 mg of PPX. In some embodiments, the capsule comprises about 1000 mg of RPPX and no more than about 0.6 mg of PPX. In some embodiments, the capsule comprises about 1000 mg of RPPX and no more than about 0.375 mg of PPX.

The modified release capsule may have a chiral purity for RPPX of at least 99.5%, preferably at least 99.6%, preferably at least 99.7%, preferably at least 99.8%, preferably at least 99.9%, preferably at least 99.95%, or more preferably at least 99.99%. In some embodiments, the chiral purity for RPPX is 100%. In some embodiments, the capsule has a chiral purity for RPPX of 99.9% or greater. In some embodiments, the capsule has a chiral purity for RPPX of 99.95% or greater. In some embodiments, the capsule has a chiral purity for RPPX of 99.99% or greater.

In a further aspect, the present invention provides a modified release pharmaceutical composition comprising at least about 25 mg of RPPX and less than about 4.5 dopaminergic activity equivalents (“DAE”). Table 1 shows the DAE for a 25 mg dose of RPPX as a function of a particular chiral purity of the RPPX in the dose and the comparative binding affinity ratio.

In some embodiments, the modified release pharmaceutical composition comprises less than about 1.5 dopaminergic activity equivalents (DAE). In some embodiments, the modified release pharmaceutical composition comprises less than about 0.15 dopaminergic activity equivalents. These DAE values are derived from the no observable adverse effect levels of RPPX as discussed herein. In some embodiments, the composition has a DAE which is less than the DAE as calculated from the MTD amount or non-effective dose amounts of PPX. With reference to non-effective dose amounts of PPX, in some embodiments, the DAE does not exceed about 1.0, does not exceed about 0.75, does not exceed about 0.5, does not exceed about 0.25, or does not exceed about 0.125. With reference to MTD amount, the modified release composition may have a DAE of below 4.5, below 0.9, or below 0.6.

In some embodiments, the modified release pharmaceutical composition comprises at least about 50 mg of RPPX. In some embodiments, the modified release pharmaceutical composition comprises at least about 75 mg of RPPX. In some embodiments, the modified release pharmaceutical composition comprises at least about 125 mg of RPPX. In some embodiments, the modified release pharmaceutical composition comprises at least about 150 mg of RPPX. In some embodiments, the modified release pharmaceutical composition comprises at least about 200 mg of RPPX. In some embodiments, the modified release pharmaceutical composition comprises at least about 250 mg of RPPX. In some embodiments, the modified release pharmaceutical composition comprises at least about 300 mg of RPPX. In some embodiments, the pharmaceutical composition comprises at least about 400 mg of RPPX. In some embodiments, the modified release pharmaceutical composition comprises at least about 500 mg of RPPX. In some embodiments, the modified release pharmaceutical composition comprises at least about 600 mg of RPPX. In some embodiments, the modified release pharmaceutical composition comprises at least about 750 mg of RPPX. In some embodiments, the modified release pharmaceutical composition comprises at least about 1000 mg of RPPX.

In some embodiments, the modified release composition is suitable for oral administration. In some embodiments, the composition is a solid oral dosage form. In some embodiments, the pharmaceutical composition is a tablet. In some embodiments, the pharmaceutical composition is a capsule.

In some embodiments, the amount of RPPX may be from about 0.01 mg/kg/day to about 10,000 mg/kg/day, from about 1 mg/kg/day to about 1,000 mg/kg/day, from about 0.1 mg/kg/day to about 1,000 mg/kg/day, from about 1 mg/kg/day to about 1,000 mg/kg/day, from about 1,000 mg/kg/day to about 10,000 mg/kg/day, or from about 1 mg/kg/day to about 100 mg/kg/day. In some embodiments, the amount of RPPX may be from about 3 mg/kg/day to about 70 mg/kg/day. In some embodiments, the amount of RPPX may be from about 7 mg/kg/day to about 40 mg/kg/day. In some embodiment, the amount of RPPX may be from about 3 mg/kg/day to about 50 mg/kg/day. In some embodiments, the dosage may be 10 mg/day to 1,500 mg/day, more preferably 1100 mg/day to 600 mg/day. The amount of RPPX in the modified release compositions may preferably be about 50 mg to about 5,000 mg, from about 100 mg to about 3,000 mg, from about 300 mg to about 1,500 mg, from about 500 mg to about 1,000 mg. In some embodiments, the amount of RPPX in the modified release compositions may be from about 50 mg to about 5,000 mg, from about 100 mg to about 5,000 mg, from about 200 mg to about 5,000 mg, from about 250 mg to about 5,000 mg, from about 300 mg to about 5,000 mg, from about 400 mg to about 5,000 mg, from 450 mg to about 5,000 mg, from about 200 mg, to about 3,000 mg, from about 250 mg to about 3,000 mg, from about 300 mg to about 3,000 mg, from about 400 mg to about 3,000 mg, from 450 mg to about 3,000 mg, from about 100 mg to about 1,000 mg, from about 200 mg to about 1,000 mg, from about 250 mg to about 1,000 mg, from about 300 mg to about 1,000 mg, from about 400 mg to about 1,000 mg, from about 600 mg to about 1,000 mg, or from 450 mg to about 1,000 mg. In some embodiments, the amount of RPPX is from about 600 mg to about 900 mg. In some embodiments, the amount of RPPX is from about 50 mg to about 5000 mg. In some embodiments, the amount of RPPX is from about 100 mg to about 3000 mg. In some embodiments, the amount of RPPX is from about 300 mg to about 1500 mg. In some embodiments, the amount of RPPX is from about 500 mg to about 1000 mg. In some embodiments, the composition is suitable for oral administration. In some embodiments, the composition is a solid oral dosage form.

The modified release composition may have a chiral purity for RPPX of at least 99.5%, preferably at least 99.6%, preferably at least 99.7%, preferably at least 99.8%, preferably at least 99.9%, preferably at least 99.95%, or more preferably at least 99.99%. In some embodiments, the chiral purity for RPPX is 100%. In some embodiments, the modified release composition has a chiral purity for RPPX of 99.9% or greater. In some embodiments, the modified release composition has a chiral purity for RPPX of 99.95% or greater. In some embodiments, the modified release composition has a chiral purity for RPPX of 99.99% or greater.

In another aspect, the present invention provides a starting daily dose of RPPX of at least about 25 mg of RPPX. In some embodiments, the starting daily dose comprises at least about 50 mg of RPPX. In some embodiments, the starting daily dose comprises at least about 75 mg of RPPX. In some embodiments, the starting daily dose comprises at least about 125 mg of RPPX. In some embodiments, the starting daily dose comprises at least about 150 mg of RPPX. In some embodiments, the starting daily dose comprises at least about 200 mg of RPPX. In some embodiments, the starting daily dose comprises at least about 300 mg of RPPX. In some embodiments, the starting daily dose comprises at least about 400 mg of RPPX. In some embodiments, the starting daily dose comprises at least about 500 mg of RPPX. In some embodiments, the starting daily dose comprises at least about 600 mg of RPPX. In some embodiments, the starting daily dose comprises at least about 750 mg of RPPX. In some embodiments, the starting daily dose comprises at least about 1000 mg of RPPX. In some embodiments, the starting daily dose comprises from about 600 mg to about 900 mg of RPPX.

In some embodiments, the starting daily dose amount of RPPX may be from about 0.01 mg/kg/day to about 10,000 mg/kg/day, from about 1 mg/kg/day to about 1,000 mg/kg/day, from about 0.1 mg/kg/day to about 1,000 mg/kg/day, from about 1 mg/kg/day to about 1,000 mg/kg/day, from about 1,000 mg/kg/day to about 10,000 mg/kg/day, or from about 1 mg/kg/day to about 1100 mg/kg/day. In some embodiments, the starting daily dose amount of RPPX may be from about 3 mg/kg/day to about 70 mg/kg/day. In some embodiments, the starting daily dose amount of RPPX may be from about 7 mg/kg/day to about 40 mg/kg/day. In some embodiment, the starting daily dose amount of RPPX may be from about 3 mg/kg/day to about 50 mg/kg/day. In some embodiments, the starting daily dose amount may be 10 mg/day to 1,500 mg/day, more preferably 100 mg/day to 600 mg/day. The starting daily dose amount of RPPX in the modified release compositions may preferably be about 50 mg to about 5,000 mg, from about 100 mg to about 3,000 mg, from about 300 mg to about 1,500 mg, from about 500 mg to about 1,000 mg. In some embodiments, the starting daily dose amount of RPPX in the modified release compositions may be about from about 25 mg to about 5,000 mg, from about 50 mg to about 5,000 mg, from about 100 mg to about 5,000 mg, from about 200 mg to about 5,000 mg, from about 250 mg to about 5,000 mg, from about 300 mg to about 5,000 mg, from about 400 mg to about 5,000 mg, from 450 mg to about 5,000 mg, from about 200 mg, to about 3,000 mg, from about 250 mg to about 3,000 mg, from about 300 mg to about 3,000 mg, from about 400 mg to about 3,000 mg, from 450 mg to about 3,000 mg, from about 100 mg to about 1,000 mg, from about 200 mg to about 1,000 mg, from about 250 mg to about 1,000 mg, from about 300 mg to about 1,000 mg, from about 400 mg to about 1,000 mg, from about 600 mg to about 1,000 mg, or from 450 mg to about 1,000 mg. In some embodiments, the starting daily dose amount of RPPX is from about 600 mg to about 900 mg. In some embodiments, the starting daily dose amount of RPPX is from about 50 mg to about 5000 mg. In some embodiments, the starting daily dose amount of RPPX is from about 100 mg to about 3000 mg. In some embodiments, the starting daily dose amount of RPPX is from about 300 mg to about 1500 mg. In some embodiments, the starting daily dose amount of RPPX is from about 500 mg to about 1000 mg.

The modified release composition may have a chiral purity for RPPX of at least 99.5%, preferably at least 99.6%, preferably at least 99.7%, preferably at least 99.8%, preferably at least 99.9%, preferably at least 99.95%, or more preferably at least 99.99%. In some embodiments, the chiral purity for RPPX is 100%. In some embodiments, the composition has a chiral purity for RPPX of 99.9% or greater. In some embodiments, the composition has a chiral purity for RPPX of 99.95% or greater. In some embodiments, the composition has a chiral purity for RPPX of 99.99% or greater.

In some embodiments, the modified release composition is suitable for oral administration. In some embodiments, the composition is a solid oral dosage form. In some embodiments, the pharmaceutical composition is a tablet. In some embodiments, the pharmaceutical composition is a capsule.

In another aspect, the present invention provides a modified release pharmaceutical formulation comprising microcrystalline cellulose in an amount from about 20% to about 50% by weight of said formulation; mannitol in about from about 10% to about 30% by weight of said formulation; crospovidone in an amount from about 2% to about 6% of said formulation; magnesium stearate in an amount from about 0.01% to about 2% of said composition; and RPPX. In some embodiments, the modified release pharmaceutical composition comprises a diluent in an amount from about 20% to about 50% by weight of said formulation; optionally, a second diluent in an amount from about 10% to about 30% by weight of said formulation; optionally, a disintegrant in an amount from about 2% to about 6% of said formulation; optionally, a lubricant in an amount from about 0.01% to about 2% of said composition; and RPPX. In some embodiments, the modified release pharmaceutical composition comprises microcrystalline cellulose, mannitol, croscarmellose sodium, magnesium stearate, or combination thereof. In some embodiments, the pharmaceutically acceptable carrier comprises microcrystalline cellulose, mannitol or combination thereof; and further optionally comprises croscarmellose sodium or magnesium stearate, or combination thereof.

The modified release formulation may have a chiral purity for RPPX of at least 99.5%, preferably at least 99.6%, preferably at least 99.7%, preferably at least 99.8%, preferably at least 99.9%, preferably at least 99.95%, or more preferably at least 99.99%. In some embodiments, the chiral purity for RPPX is 100%. In some embodiments, the formulation has a chiral purity for RPPX of 99.9% or greater. In some embodiments, the formulation has a chiral purity for RPPX of 99.95% or greater. In some embodiments, the formulation has a chiral purity for RPPX of 99.99% or greater.

The amount of RPPX in the modified release formulation may preferably be about 50 mg to about 5,000 mg, from about 100 mg to about 3,000 mg, from about 300 mg to about 1,500 mg, from about 500 mg to about 1,000 mg. In some embodiments, the starting daily dose amount of RPPX in the formulation may be from about 50 mg to about 5,000 mg, from about 100 mg to about 5,000 mg, from about 200 mg to about 5,000 mg, from about 250 mg to about 5,000 mg, from about 300 mg to about 5,000 mg, from about 400 mg to about 5,000 mg, from 450 mg to about 5,000 mg, from about 200 mg, to about 3,000 mg, from about 250 mg to about 3,000 mg, from about 300 mg to about 3,000 mg, from about 400 mg to about 3,000 mg, from 450 mg to about 3,000 mg, from about 100 mg to about 1,000 mg, from about 200 mg to about 1,000 mg, from about 250 mg to about 1,000 mg, from about 300 mg to about 1,000 mg, from about 400 mg to about 1,000 mg, from about 600 mg to about 1,000 mg, or from 450 mg to about 1,000 mg. In some embodiments, the formulation of RPPX is from about 600 mg to about 900 mg.

In some embodiments, the present invention provides a modified release pharmaceutical composition comprising a pharmaceutical composition comprising microcrystalline cellulose in an amount from about 20% to about 50% by weight of said composition; mannitol in an amount from about 10% to about 30% by weight of said composition; crospovidone in an amount from about 2% to about 6% of said composition; magnesium stearate in an amount from about 0.01% to about 2% of said composition; and RPPX. In some embodiments, the modified release composition is suitable for oral administration. In some embodiments, the composition is a solid oral dosage form.

The modified release composition may have a chiral purity for RPPX of at least 99.5%, preferably at least 99.6%, preferably at least 99.7%, preferably at least 99.8%, preferably at least 99.9%, preferably at least 99.95%, or more preferably at least 99.99%. In some embodiments, the chiral purity for RPPX is 100%. In some embodiments, the composition has a chiral purity for RPPX of 99.9% or greater. In some embodiments, the composition has a chiral purity for RPPX of 99.95% or greater. In some embodiments, the composition has a chiral purity for RPPX of 99.99% or greater.

The amount of RPPX in the modified release compositions may preferably be about 50 mg to about 5,000 mg, from about 100 mg to about 3,000 mg, from about 300 mg to about 1,500 mg, from about 500 mg to about 1,000 mg. In some embodiments, the starting daily dose amount of RPPX in the modified release compositions may be about from about 25 mg to about 5,000 mg, from about 50 mg to about 5,000 mg, from about 100 mg to about 5,000 mg, from about 200 mg to about 5,000 mg, from about 250 mg to about 5,000 mg, from about 300 mg to about 5,000 mg, from about 400 mg to about 5,000 mg, from 450 mg to about 5,000 mg, from about 200 mg, to about 3,000 mg, from about 250 mg to about 3,000 mg, from about 300 mg to about 3,000 mg, from about 400 mg to about 3,000 mg, from 450 mg to about 3,000 mg, from about 100 mg to about 1,000 mg, from about 200 mg to about 1,000 mg, from about 250 mg to about 1,000 mg, from about 300 mg to about 1,000 mg, from about 400 mg to about 1,000 mg, from about 600 mg to about 1,000 mg, or from 450 mg to about 1,000 mg. In some embodiments, the amount of RPPX is from about 600 mg to about 900 mg. In some embodiments, the present invention further provides modified release pharmaceutical compositions comprising RPPX having about 25 neuroprotective activity equivalents and less than about 4.5 dopaminergic activity equivalents. In some embodiments, the modified release pharmaceutical composition has less than about 1.5 dopaminergic activity equivalents. In some embodiments, the modified release pharmaceutical composition has less than about 0.15 dopaminergic activity equivalents.

In some embodiments, the modified release pharmaceutical composition has at least about 50, at least about 75, at least about 125, at least about 150, at least about 200, at least about 300, at least about 400, at least about 500, at least about 750, at least about 750, or at least about 100 neuroprotective activity equivalents. In some embodiments, the modified release pharmaceutical composition has from about 50 to about 5,000, from about 100 to about 3,000, from about 300 to about 1,500, from about 500 to about 1,000, from about 25 to about 5,000, from about 100 to about 5,000, from about 200 to about 5,000, from about 250 to about 5,000, from about 300 to about 5,000, from about 400 to about 5,000, from 450 to about 5,000, from about 200, to about 3,000, from about 250 to about 3,000, from about 300 to about 3,000, from about 400 to about 3,000, from 450 to about 3,000, from about 100 to about 1,000, from about 200 to about 1,000, from about 250 to about 1,000, from about 300 to about 1,000, from about 400 to about 1,000, from about 600 to about 1,000, from 450 to about 1,000, or from about 600 to about 900 neuroprotective activity equivalents.

In some embodiments, the modified release pharmaceutical composition has from about 50 to about 5,000 neuroprotective activity equivalents; and less than from about 1.5 dopaminergic activity equivalents. In some embodiments, the pharmaceutical composition has about 100 to about 3,000 neuroprotective activity equivalents; and less than from about 1.5 dopaminergic activity equivalents. In some embodiments, the pharmaceutical composition has about 200 to about 3,000 neuroprotective activity equivalents; and less than from about 1.5 dopaminergic activity equivalents. In some embodiments, the pharmaceutical composition has about 300 to about 1,500 neuroprotective activity equivalents; and less than from about 1.5 dopaminergic activity equivalents. In some embodiments, the pharmaceutical composition has about 500 to about 1,000 neuroprotective activity equivalents; and less than from about 1.5 dopaminergic activity equivalents. In some embodiments, the pharmaceutical composition has about 50 to about 5,000 neuroprotective activity equivalents; and less than from about 0.15 dopaminergic activity equivalents. In some embodiments, the pharmaceutical composition has about 100 to about 3,000 neuroprotective activity equivalents; and less than from about 0.15 dopaminergic activity equivalents. In some embodiments, the pharmaceutical composition has about 200 to about 3,000 neuroprotective activity equivalents; and less than from about 0.15 dopaminergic activity equivalents. In some embodiments, the pharmaceutical composition has about 300 to about 1,500 neuroprotective activity equivalents; and less than from about 0.15 dopaminergic activity equivalents. In some embodiments, the pharmaceutical composition has about 500 to about 1,000 neuroprotective activity equivalents; and less than from about 0.15 dopaminergic activity equivalents.

In some embodiments, the modified release pharmaceutical composition is a solid oral dosage form. In some embodiments, the pharmaceutical composition is a tablet. In some embodiments, the pharmaceutical composition is a capsule.

Extended Release Formulations. Many active pharmaceutical agents, including drugs and prodrugs, have been formulated as orally deliverable dosage forms providing sustained release (otherwise known as slow release or extended release) of such agents over a period of time effective to permit once daily administration. A well-known system for formulating such dosage forms involves a matrix comprising a hydrophilic polymer wherein the agent is dispersed; the agent is released over a period of time in the gastrointestinal tract upon dissolution or erosion of the matrix. Sustained-release dosage forms comprising such a matrix system are conveniently prepared as compressed tablets, often described as “matrix tablets”.

Drugs and prodrugs having relatively high solubility in water, for example a solubility of about 10 mg/ml or greater, present challenges to the formulator wishing to provide a sustained-release dosage form, and the higher the solubility the greater are the challenges. These challenges are well illustrated in the cases of pramipexole dihydrochloride, which has a solubility in water of about 200 mg/ml.

A three times daily dosing regimen for immediate-release pramipexole dihydrochloride tablets is well tolerated, but patient compliance would be much improved if a once-daily regimen were possible.

The extended release formulations are generally prepared as diffusion or osmotic systems, for example, as described in “Remington—The Science and Practice of Pharmacy” (20th ed., Lippincott Williams & Wilkins, Baltimore, Md., 2000). A diffusion system typically consists of two types of devices, reservoir devices and matrix devices, both of which are well known and described in the art. The matrix devices are generally prepared by compressing the drug with a slowly dissolving polymer carrier into a tablet form. The three major types of materials used in the preparation of matrix devices are insoluble plastics, hydrophilic polymers, and fatty compounds. Plastic matrices include, but are not limited to, methyl acrylate-co-methyl methacrylate, polyvinyl chloride, and polyethylene. Hydrophilic polymers include, but are not limited to, methylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and CARBOPOL 934, and polyethylene oxides. Fatty compounds include, but are not limited to, various waxes such as carnauba wax and glyceryl tristearate.

Alternatively, extended release formulations can be prepared using osmotic systems or by applying a semi-permeable coating to the dosage form. In the latter case, the desired drug release profile can be achieved by combining low permeable and high permeable coating materials in suitable proportion.

One exemplary extended release formulation that may be used in certain embodiments of modified release formulations of RPPX of the present invention is disclosed in U.S. Publication No. 20050226926 entitled “Sustained-release tablet composition of pramipexole,” herein incorporated by reference in its entirety, which describes sustained-release pharmaceutical compositions in a form of an orally deliverable tablet that comprises a water-soluble salt of PPX, dispersed in a matrix comprising a hydrophilic polymer and a starch having a tensile strength of at least about 0.15 kN cm⁻² at a solid fraction representative of the tablet. Another exemplary extended release formulation that may be used in certain embodiments of modified release formulations of RPPX of the present invention is disclosed in U.S. Publication No. 20060051419 entitled “Extended release pellet formulation containing pramipexole or a pharmaceutically acceptable salt thereof,” herein incorporated by reference in its entirety, which describes extended release pellet formulations containing PPX or a pharmaceutically acceptable salt thereof, method for manufacturing the same and use thereof.

It is believed to be particularly desirable to provide sustained-release oral dosage forms that provide drug release at a substantially constant release rate over an extended time period. In this manner, for many drugs, the plasma drug concentration initially ascends for a short period of time as drug release begins and then remains substantially constant over an extended time period as drug release continues at a constant rate. For many drugs, this substantially constant plasma drug concentration correlates with substantially constant drug effectiveness over a prolonged therapy period. In addition, because an initial relatively high peak plasma drug concentration is avoided, side effects may be less of a problem. Accordingly, advantages of constant-release dosage forms include decreasing the number of doses of a drug that need to be administered over time and providing a better balance of desired and undesired pharmacological effects of the drug.

Osmotic dosage forms, in particular, have been notably successful at providing constant-release of drugs over extended time periods. Osmotic dosage forms, in general, utilize osmotic pressure to generate a driving force for imbibing fluid into a compartment formed, at least in part, by a semipermeable wall that permits free diffusion of fluid but not drug or osmotic agent(s), if present. A substantially constant rate of drug release can be achieved by designing the system to provide a relatively constant osmotic pressure and having suitable exit means for the drug formulation to permit the drug formulation to be released at a rate that corresponds to the rate of fluid imbibed as a result of the relatively constant osmotic pressure. A significant advantage to osmotic systems is that operation is pH-independent and thus continues at the osmotically-determined rate throughout an extended time period even as the dosage form transits the gastrointestinal tract and encounters differing microenvironments having significantly different pH values.

Surprisingly simple but highly effective osmotic devices comprising drug in a mixture with excipients, optionally including osmotically active component(s), within the compartment are known in the art. Although effective for many drugs, the release rate in these devices often declines over time and complete delivery of the drug load may not occur. A more sophisticated type of osmotic device comprises two component layers within the compartment formed by the semipermeable wall. One component layer comprises drug in a mixture with excipients, optionally including osmotically active component(s), that will form a deliverable drug formulation within the compartment and the second component layer comprises osmotically active component(s) but does not contain drug. The osmotically active component(s) in the second component layer typically comprise osmopolymer(s) having relatively large molecular weights and which exhibit “swelling” as fluid is imbibed such that release of these components through the drug formulation exit means does not occur. The second component layer is referred to as a “push” layer since, as fluid is imbibed, the osmopolymer(s) swell and push against the deliverable drug formulation of the first component layer to thereby facilitate release of the drug formulation at a substantially constant rate. The above-described devices are known, for example, from the following US patents, owned by Alza Corporation: U.S. Pat. Nos. 4,327,725; 4,612,008; 4,783,337; and 5,082,668, each of which is incorporated in its entirety by reference herein. Other suitable formulations that employ, for example, the Oros™ sustained release technology platform of Alza Corporation are described in, for example, U.S. Pat. Nos. 6,919,373; 6,930,129; 5,024,843; 5,091,190; 5,545,413; 5,591,454; 5,674,895; 5,840,754; 5,912,268; 6,262,115; and 6,919,092, which may be useful in the modified release formulations of RPPX.

The devices with different drug release mechanisms described above can be combined in a final dosage form comprising single or multiple units. Examples of multiple units include multilayer tablets and capsules containing tablets, beads, granules, etc.

An immediate release portion can be added to the extended release system by means of either applying an immediate release layer on top of the extended release core using coating or compression processes or in a multiple unit system, such as a capsule, containing extended and immediate release beads.

Extended release tablets containing hydrophilic polymers are prepared by techniques commonly known in the art such as direct compression, wet granulation, or dry granulation processes. Their formulations usually incorporate polymers, diluents, binders, and lubricants as well as the active pharmaceutical ingredient. The usual diluents include inert powdered substances such as any of many different kinds of starch, powdered cellulose, especially crystalline and microcrystalline cellulose, sugars such as fructose, mannitol and sucrose, grain flours and similar edible powders. Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts such as sodium chloride and powdered sugar. Powdered cellulose derivatives are also useful. Typical tablet binders include substances such as starch, gelatin and sugars such as lactose, fructose, and glucose. Natural and synthetic gums, including acacia, alginates, methylcellulose, and polyvinylpyrrolidine can also be used. Polyethylene glycol, hydrophilic polymers, ethylcellulose and waxes can also serve as binders. A lubricant is necessary in a tablet formulation to prevent the tablet and punches from sticking in the die. The lubricant is chosen from such slippery solids as talc, magnesium and calcium stearate, stearic acid and hydrogenated vegetable oils.

Extended release tablets containing wax materials are generally prepared using methods known in the art such as a direct blend method, a congealing method, and an aqueous dispersion method. In a congealing method, the drug is mixed with a wax material and either spray-congealed or congealed, screened, and processed.

Modified or Extended Release Transdermal Formulations. Transdermal dosage forms are particularly useful for timed-release and sustained-release of active agents. Transdermal systems for delivering a wide variety of drugs or other beneficial agents are described in U.S. Pat. Nos. 3,598,122; 3,598,123; 3,731,683; 3,797,494; 4,031,894; 4,144,317; 4,201,211; 4,286,592; 4,314,557; 4,379,454; 4,435,180; 4,559,222; 4,568,343; 4,573,995; 4,588,580; 4,645,502; 4,698,062; 4,704,282; 4,725,272; 4,781,924; 4,788,062; 4,816,258; 4,849,226; 4,904,475; 4,908,027; 4,917,895; 4,938,759; 4,943,435; 5,004,610; 5,071,656; 5,122,382; 5,141,750; 5,284,660; 5,314,694; 5,342,623; 5,411,740; 5,635,203, 20080026043; and 20080020028—hereby incorporated in their entirety by reference. The most common transdermal dosage form is a diffusion-driven transdermal system (particularly in the form of a patch) using either a fluid reservoir or a drug-in-adhesive matrix system. Other transdermal dosage forms include, but are not limited to, topical gels, lotions, ointments, transmucosal systems and devices, and iontophoretic (electrical-diffusion) delivery systems. See, for example, U.S. Pat. No. 4,626,539 to Aungst et al., which discloses a pharmaceutical composition purportedly useful for transdermal delivery of an opioid to a mammalian circulation system; U.S. Pat. No. 4,806,341 to Chien et al., which discloses transdermal-absorption dosage units comprising a backing layer, an adhesive polymer layer, and an adjoining layer of a solid polymer matrix containing a morphinan narcotic analgesic or antagonist and skin penetration enhancers; and U.S. Pat. No. 5,069,909 to Sharma et al., which discloses methods for sustained administration of buprenorphine using transdermal delivery from a laminated composite patch. U.S. Pat. No. 5,830,497 to Yamanaka et al. discloses a medicated plaster composition as a dosage form for percutaneous absorption, the composition containing either (1) a basic drug and an acidic substance; (2) a salt of a basic drug and a basic substance; or (3) a basic drug and a salt of a basic drug. U.S. Pat. No. 5,804,215 to Cubbage et al. discloses a disposal system for a transdermal patch containing a medicament comprising an adhesive coated, flexible, tear-resistant substrate. The used transdermal patch allegedly adheres to the substrate in order to encapsulate and deter access to the transdermal patch. Rubber-based adhesives are disclosed as being preferred.

The modified release formulations may also be administered adjunctively with other active compounds such as analgesics, anti-inflammatory drugs, antipyretics, antidepressants, antiepileptics, antihistamines, antimigraine drugs, antimuscarinics, anxiolytics, sedatives, hypnotics, antipsychotics, bronchodilators, anti asthma drugs, cardiovascular drugs, corticosteroids, dopaminergics or other dopamine agonists, including but not limited to Requip IR (ropinirole), Stalevo (carbidopa, levodopa, entacapone), COMtan (entacapone), Eldepryyl (selegiline), Neupro (rotigotine), and Azilect (rasagaline), electrolytes, gastrointestinal drugs, muscle relaxants, nutritional agents, vitamins, parasympathomimetics, stimulants, anorectics and anti-narcoleptics. The compounds of the present invention can also be administered in combination with other active ingredients, such as, for example, adjuvants, protease inhibitors, or other compatible drugs or compounds where such combination is seen to be desirable or advantageous in achieving the desired effects of the methods described herein. By adjunctive administration is meant simultaneous administration of the compounds, in the same dosage form, simultaneous administration in separate dosage forms, and separate administration of the compounds.

Methods of Manufacturing Modified Release Formulations

As will be appreciated by those skilled in the art and as described in the pertinent texts and literature, a number of methods are available for preparing drug-containing tablets, beads, granules or particles that provide a variety of drug release profiles. Such methods include, but are not limited to, the following: coating a drug or drug-containing composition with an appropriate coating material, typically although not necessarily incorporating a polymeric material, increasing drug particle size, placing the drug within a matrix, and forming complexes of the drug with a suitable complexing agent.

A preferred method for preparing extended release tablets is by compressing a drug-containing blend, e.g., blend of granules, prepared using a direct blend, wet-granulation, or dry-granulation process. Extended release tablets may also be molded rather than compressed, starting with a moist material containing a suitable water-soluble lubricant. However, tablets are preferably manufactured using compression rather than molding. A preferred method for forming extended release drug-containing blend is to mix drug particles directly with one or more excipients such as diluents (or fillers), binders, disintegrants, lubricants, glidants, and colorants. As an alternative to direct blending, a drug-containing blend may be prepared by using wet-granulation or dry-granulation processes. Beads containing the active agent may also be prepared by any one of a number of conventional techniques, typically starting from a fluid dispersion. For example, a typical method for preparing drug-containing beads involves dispersing or dissolving the active agent in a coating suspension or solution containing pharmaceutical excipients such as polyvinylpyrrolidone, methylcellulose, talc, metallic stearates, silicone dioxide, plasticizers or the like. The admixture is used to coat a bead core such as a sugar sphere (or so-called “non-pareil”) having a size of approximately 60 to 20 mesh.

An alternative procedure for preparing drug beads is by blending drug with one or more pharmaceutically acceptable excipients, such as microcrystalline cellulose, lactose, cellulose, polyvinyl pyrrolidone, talc, magnesium stearate, a disintegrant, etc., extruding the blend, spheronizing the extrudate, drying and optionally coating to form the immediate release beads.

Methods of Treatment, Uses, and Compositions and Compounds for Use

In another aspect, the present invention provides a method for treating a neurodegenerative disease by administering a therapeutically effective amount of RPPX. In accordance with this embodiment, the RPPX may be formulated as a modified release pharmaceutical or therapeutic composition by combining with one or more pharmaceutically acceptable carriers. Embodiments include modified release pharmaceutical or therapeutic compositions that may be administered orally, preferably as a solid oral dose, and more preferably as a solid oral dose that may be a capsule or tablet. In a preferred embodiment, the modified release pharmaceutical or therapeutic composition is formulated in tablet or capsule form for use in oral administration routes. The compositions and amounts of non-active ingredients in such a formulation may depend on the amount of the active ingredient, and on the size and shape of the tablet or capsule. Such parameters may be readily appreciated and understood by one of skill in the art. The therapeutically effective amount of RPPX may be effective as an inhibitor of oxidative stress, an inhibitor of lipid peroxidation or in detoxification of oxygen radicals, or as an inhibitor of cell death pathways.

In some embodiments, the therapeutically effective amount of RPPX may be from about 0.01 mg/kg/day to about 10,000 mg/kg/day, from about 1 mg/kg/day to about 1,000 mg/kg/day, from about 0.1 mg/kg/day to about 1,000 mg/kg/day, from about 1 mg/kg/day to about 1,000 mg/kg/day, from about 1,000 mg/kg/day to about 10,000 mg/kg/day, or from about 1 mg/kg/day to about 100 mg/kg/day. In some embodiments, the therapeutically effective amount of RPPX may be from about 3 mg/kg/day to about 70 mg/kg/day. In some embodiments, the therapeutically effective amount of RPPX may be from about 7 mg/kg/day to about 40 mg/kg/day. In some embodiment, the therapeutically effective amount of RPPX may be from about 3 mg/kg/day to about 50 mg/kg/day. In some embodiments, the dosage may be 10 mg/day to 1,500 mg/day, more preferably 100 mg/day to 600 mg/day. In some embodiments, the therapeutically effective amount of RPPX may be from about 50 mg to about 5,000 mg, from about 100 mg to about 3,000 mg, preferably from about 300 mg to about 1,500 mg, or more preferably from about 500 mg to about 1,000 mg. In some embodiments, the therapeutically effective amount of RPPX may be from about 50 mg to about 5,000 mg, from about 100 mg to about 5,000 mg, from about 200 mg to about 5,000 mg, from about 250 mg to about 5,000 mg, from about 300 mg to about 5,000 mg, from about 400 mg to about 5,000 mg, from 450 mg to about 5,000 mg, from about 200 mg to about 3,000 mg, from about 250 mg to about 3,000 mg, from about 300 mg to about 3,000 mg, from about 400 mg to about 3,000 mg, from 450 mg to about 3,000 mg, from about 100 mg to about 1,000 mg, from about 200 mg to about 1,000 mg, from about 250 mg to about 1,000 mg, from about 300 mg to about 1,000 mg, from about 400 mg to about 1,000 mg, from about 600 mg to about 1,000 mg, or from 450 mg to about 1,000 mg. In some embodiments, the therapeutically effective amount of RPPX is from about 600 mg to about 900 mg.

The embodiment may have a chiral purity for RPPX of at least 99.5%, preferably at least 99.6%, preferably at least 99.7%, preferably at least 99.8%, preferably at least 99.9%, preferably at least 99.95% and more preferably at least 99.99%. In a preferred embodiment, the chiral purity for the R(+) enantiomer of pramipexole may be 100%.

In a further aspect, the present invention further provides a method of treating an acute neurodegenerative disease in a patient in need thereof, comprising administering to the patient a daily dose amount of about 50 mg to about 5,000 mg of RPPX. In some embodiments, the present invention provides use of a daily dose amount of about 50 mg to about 5,000 mg of RPPX for the preparation of medicament for use in a method of treatment of an acute neurodegenerative disorder in a patient. In another aspect, the present invention provides a daily dose amount of about 50 mg to about 5,000 mg of RPPX for use of in method of treatment of an acute neurodegenerative disorder in a patient.

In some embodiments, the acute neurodegenerative disease is selected from stroke, neurotrauma, acute metabolic dysfunction, sequelae from cerebral seizure, status epilepticus, and acute encephalitis.

In some embodiments, the patient is a naïve patient.

In some embodiments, the daily dose amount of RPPX may be from about 0.01 mg/kg/day to about 10,000 mg/kg/day, from about 1 mg/kg/day to about 1.000 mg/kg/day, from about 0.1 mg/kg/day to about 1,000 mg/kg/day, from about 1 mg/kg/day to about 1,000 mg/kg/day, from about 1,000 mg/kg/day to about 10,000 mg/kg/day, or from about 1 mg/kg/day to about 100 mg/kg/day. In some embodiments, the daily dose amount of RPPX may be from about 3 mg/kg/day to about 70 mg/kg/day. In some embodiments, the daily dose amount of RPPX may be from about 7 mg/kg/day to about 40 mg/kg/day. In some embodiment, the daily dose amount of RPPX may be from about 3 mg/kg/day to about 50 mg/kg/day. In some embodiments, the daily dose amount may be 10 mg/day to 1,500 mg/day, more preferably 100 mg/day to 600 mg/day. In some embodiments, the daily dose amount of RPPX is from about 50 mg to about 5,000 mg, from about 100 mg to about 3,000 mg, from about 300 mg to about 1,500 mg, or from about 500 mg to about 1,000 mg. In some embodiments, the daily dose amount of RPPX is from about 50 mg to about 5,000 mg, from about 100 mg to about 5,000 mg, from about 200 mg to about 5,000 mg, from about 250 mg to about 5,000 mg, from about 300 mg to about 5,000 mg, from about 400 mg to about 5,000 mg, from 450 mg to about 5,000 mg, from about 200 mg to about 3,000 mg, from about 250 mg to about 3,000 mg, from about 300 mg to about 3,000 mg, from about 400 mg to about 3,000 mg, from 450 mg to about 3,000 mg, from about 100 mg to about 1,000 mg, from about 200 mg to about 1,000 mg, from about 250 mg to about 1,000 mg, from about 300 mg to about 1,000 mg, from about 400 mg to about 1,000 mg, from about 600 mg to about 1,000 mg, or from 450 mg to about 1,000 mg. In some embodiments, the daily dose amount of RPPX is from about 600 mg to about 900 mg. In some embodiments, the daily dose amount is from about 500 mg to about 1,000 mg of RPPX. In some embodiments, daily dose amount is from about 50 mg to about 5,000 mg of RPPX. In some embodiments, the daily dose amount is from about 100 mg to about 3,000 mg of RPPX. In some embodiments, daily dose amount is from about 200 mg to about 3,000 mg of RPPX. In some embodiments, daily dose amount is from about 300 mg to about 1,500 mg of RPPX. In some embodiments, daily dose amount is from about 500 mg to about 1,000 mg of RPPX.

In some embodiments, the chiral purity for RPPX is 99.5%, or greater. In some embodiments, the chiral purity for RPPX is 99.6%, or greater. In some embodiments, the chiral purity for RPPX is 99.7%, or greater. In some embodiments, the chiral purity for RPPX is 99.8%, or greater. In some embodiments, the chiral purity for the RPPX is 99.9%, or greater. In some embodiments, the chiral purity for the RPPX is 99.95%, or greater. In some embodiments, the chiral purity for the RPPX is 99.99% or greater.

In some embodiments, the daily dose amount further comprises a no observable adverse effect level amount of PPX. In some embodiments, the no observable effective dose amount of PPX is an below 4.5 mg, below 1.5 mg, or below 0.15 mg per day.

In some embodiments, the daily dose amount further comprises a non-effective dose amount of PPX. In some embodiments, the non-effective dose amount of PPX is an amount that does not exceed a total dose of 1.0 mg per day. In some embodiments, the non-effective dose amount of PPX is an amount that does not exceed a total dose of 0.75 mg/day, 0.5 mg/day, 0.25 mg/day, or 0.125 mg/day. In some embodiments, the non-effective dose mount of PPX does not exceed a total dose of 0.125 mg/day.

In some embodiments of the methods of the invention, the daily dose amount is about 100 mg to about 3,000 mg of RPPX and the chiral purity for the RPPX is 99.95% or greater.

In some embodiments of the methods of the invention, the daily dose amount is from about 200 to about 3,000 mg of RPPX and the chiral purity for the RPPX is 99.95% or greater.

In some embodiments of the methods of the invention, the daily dose amount is from about 300 to about 1,500 mg of RPPX and the chiral purity for the RPPX is 99.95% or greater.

In some embodiments of the methods of the invention, the daily dose amount is from about 500 mg to about 1,000 mg of RPPX and the chiral purity for the RPPX is 99.95% or greater.

In some embodiments of the methods of the invention, the daily dose amount is from about 100 mg to 3,000 mg of RPPX and the daily dose amount further comprises less than about 0.05 mg of PPX.

In some embodiments of the methods of the invention, the daily dose amount is from about 200 mg to about 3,000 mg of RPPX and the daily dose amount further comprises less than about 0.05 mg of PPX.

In some embodiments of the methods of the invention, the daily dose amount is from about 300 to about 1,500 mg of RPPX and the daily dose amount further comprises less than about 0.05 mg of PPX.

In some embodiments of the methods of the invention, the daily dose amount is from about 500 mg to about 1,000 mg of RPPX and the daily dose amount further comprises less than about 0.05 mg of PPX.

In another embodiment, the RPPX in each of the method embodiments described herein is administered as a modified release pharmaceutical composition. In some embodiments, the modified release pharmaceutical composition is a tablet. In some embodiments, the modified release pharmaceutical composition is a capsule. In some embodiments, the modified release pharmaceutical composition comprises a pharmaceutically acceptable carrier.

The embodiments for disease states, patient type (naïve vs. not naïve), daily dose amounts, no observable adverse effect level dose amounts, non-effective dose amounts, and chiral purities for the methods of the invention, which are described herein separately for the sake of brevity, can be joined in any suitable combination. Any of the embodiments described herein for the methods can also be used for the uses of RPPX in the preparation of medicaments for use in methods of treating an acute neurodegenerative disease, or in the daily dose of RPPX for use in a method of treating an acute neurodegenerative disorder.

In a further aspect, the present invention further provides a method of treating a chronic neurodegenerative disease in a patient in need thereof, comprising administering to the patient a daily dose amount of about 50 mg to about 5,000 mg of RPPX. In some embodiments, the present invention provides use of a daily dose amount of about 50 mg to about 5,000 mg of RPPX for the preparation of medicament for use in a method of treatment of a chronic neurodegenerative disorder in a patient. In some embodiments, the present invention provides a daily dose amount of about 50 mg to about 5,000 mg of RPPX for use of in method of treatment of a chronic neurodegenerative disorder in a patient.

In some embodiments, the chronic neurodegenerative disease is selected from primary neurodegenerative disease, Huntington's Chorea, metabolically induced neurological damage, senile dementia of Alzheimer's type, age associated cognitive dysfunction, vascular dementia, multi-infarct dementia, Lewy body dementia, neurodegenerative dementia, neurodegenerative movement disorder, ataxia, Friedreich's ataxia, multiple sclerosis, spinal muscular atrophy, primary lateral sclerosis, seizure disorders, motor neuron disorder or disease, inflammatory demyelinating disorder, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, hepatic encephalopathy, and chronic encephalitis.

In some embodiments, the patient is a naïve patient.

In some embodiments, the daily dose amount of RPPX may be from about 0.01 mg/kg/day to about 10,000 mg/kg/day, from about 1 mg/kg/day to about 1,000 mg/kg/day, from about 0.1 mg/kg/day to about 1,000 mg/kg/day, from about 1 mg/kg/day to about 1,000 mg/kg/day, from about 1,000 mg/kg/day to about 10,000 mg/kg/day, or from about 1 mg/kg/day to about 100 mg/kg/day. In some embodiments, the daily dose amount of RPPX may be from about 3 mg/kg/day to about 70 mg/kg/day. In some embodiments, the daily dose amount of RPPX may be from about 7 mg/kg/day to about 40 mg/kg/day. In some embodiment, the daily dose amount of RPPX may be from about 3 mg/kg/day to about 50 mg/kg/day. In some embodiments, the daily dose amount may be 10 mg/day to 1,500 mg/day, more preferably 100 mg/day to 600 mg/day. In some embodiments, the daily dose amount of RPPX is from about 50 mg to about 5,000 mg, from about 100 mg to about 3,000 mg, from about 300 mg to about 1,500 mg, or from about 500 mg to about 1,000 mg. In some embodiments, the daily dose amount of RPPX is from about 50 mg to about 5,000 mg, from about 100 mg to about 5,000 mg, from about 200 mg to about 5,000 mg, from about 250 mg to about 5,000 mg, from about 300 mg to about 5,000 mg, from about 400 mg to about 5,000 mg, from 450 mg to about 5,000 mg, from about 200 mg to about 3,000 mg, from about 250 mg to about 3,000 mg, from about 300 mg to about 3,000 mg, from about 400 mg to about 3,000 mg, from 450 mg to about 3,000 mg, from about 100 mg to about 1,000 mg, from about 200 mg to about 1,000 mg, from about 250 mg to about 1,000 mg, from about 300 mg to about 1,000 mg, from about 400 mg to about 1,000 mg, from about 600 mg to about 1,000 mg, or from 450 mg to about 1.000 mg. In some embodiments, the daily dose amount of RPPX is from about 600 mg to about 900 mg. In some embodiments, the daily dose amount is from about 500 mg to about 1,000 mg of RPPX. In some embodiments, daily dose amount is from about 50 mg to about 5,000 mg of RPPX. In some embodiments, daily dose amount is from about 100 mg to about 3,000 mg of RPPX. In some embodiments, daily dose amount is from about 200 mg to about 3,000 mg of RPPX. In some embodiments, daily dose amount is from about 300 mg to about 1,500 mg of RPPX. In some embodiments, the daily dose amount is from about 500 mg to about 1,000 mg of RPPX.

In some embodiments, the chiral purity for RPPX is 99.5%, or greater. In some embodiments, the chiral purity for RPPX is 99.6%, or greater. In some embodiments, the chiral purity for RPPX is 99.7%, or greater. In some embodiments, the chiral purity for RPPX is 99.8%, or greater. In some embodiments, the chiral purity for the RPPX is 99.9%, or greater. In some embodiments, the chiral purity for the RPPX is 99.95%, or greater. In some embodiments, the chiral purity for the RPPX is 99.99% or greater.

In some embodiments, the daily dose amount further comprises a no observable adverse effect level amount of PPX. In some embodiments, the no observable effective dose amount of PPX is an below 4.5 mg, below 1.5 mg, or below 0.15 mg per day.

In some embodiments, the daily dose amount further comprises a non-effective dose amount of PPX. In some embodiments, the non-effective dose amount of PPX is an amount that does not exceed a total dose of 1.0 mg per day. In some embodiments, the non-effective dose amount of PPX is an amount that does not exceed a total dose of 0.75 mg/day, 0.5 mg/day, 0.25 mg/day, or 0.125 mg/day. In some embodiments, the non-effective dose mount of PPX does not exceed a total dose of 0.125 mg/day.

In some embodiments of the methods of the invention, the daily dose amount is about 100 mg to about 3,000 mg of RPPX and the chiral purity for the RPPX is 99.95% or greater.

In some embodiments of the methods of the invention, the daily dose amount is from about 200 to about 3,000 mg of RPPX and the chiral purity for the RPPX is 99.95% or greater.

In some embodiments of the methods of the invention, the daily dose amount is from about 300 to about 1,500 mg of RPPX and the chiral purity for the RPPX is 99.95% or greater.

In some embodiments of the methods of the invention, the daily dose amount is from about 500 mg to about 1,000 mg of RPPX and the chiral purity for the RPPX is 99.95% or greater.

In some embodiments of the methods of the invention, the daily dose amount is from about 100 mg to 3,000 mg of RPPX and the daily dose amount further comprises less than about 0.05 mg of PPX.

In some embodiments of the methods of the invention, the daily dose amount is from about 200 mg to about 3,000 mg of RPPX and the daily dose amount further comprises less than about 0.05 mg of PPX.

In some embodiments of the methods of the invention, the daily dose amount is from about 300 to about 1,500 mg of RPPX and the daily dose amount further comprises less than about 0.05 mg of PPX.

In some embodiments of the methods of the invention, the daily dose amount is from about 500 mg to about 1,000 mg of RPPX and the daily dose amount further comprises less than about 0.05 mg of PPX.

In another embodiment, the RPPX in each of the method embodiments described herein is administered as a modified release pharmaceutical composition. In some embodiments, the modified release pharmaceutical composition is a tablet. In some embodiments, the modified release pharmaceutical composition is a capsule. In some embodiments, the modified release pharmaceutical composition comprises a pharmaceutically acceptable carrier.

The embodiments for disease states, patient type (naïve vs. not naïve), daily dose amounts, no observable adverse effect level dose amounts, non-effective dose amounts, and chiral purities for the methods of the invention, which are described herein separately for the sake of brevity, can be joined in any suitable combination. Any of the embodiments described herein for the methods can also be used for the uses of RPPX in the preparation of medicaments for use in methods of treating a chronic neurodegenerative disease, or in the daily dose of RPPX for use in a method of treating a chronic neurodegenerative disorder.

Kits

In another aspect, the present invention provides a kit comprising one or more modified release pharmaceutical compositions comprising RPPX and instructions for administering or prescribing the one or more pharmaceutical compositions, comprising a direction to administer or prescribe the one or more modified release pharmaceutical compositions in an amount sufficient to result in administration of a starting daily dose of at least about 50 mg to about 5,000 mg of RPPX to a patient. In addition, in some embodiments, the present invention provides kits comprising one or more modified release pharmaceutical compositions according to any of the previous embodiments of the compositions described herein, or any combination thereof, and instructions for administering or prescribing the one or more modified release pharmaceutical compositions, comprising a direction to administer or prescribe the one or more modified release pharmaceutical compositions according to the embodiments of the methods described herein, or any combination thereof.

The kits of the invention may have a chiral purity for RPPX of at least 99.5%, preferably at least 99.6%, preferably at least 99.7%, preferably at least 99.8%, preferably at least 99.9%, preferably at least 99.95% and more preferably at least 99.99%. In a preferred embodiment, the chiral purity for the R(+) enantiomer of pramipexole may be 100%. In some embodiments, the chiral purity for the RPPX is 99.9% or greater. In some embodiments, the chiral purity for the RPPX is 99.95% or greater. In some embodiments, the chiral purity for the RPPX is 99.99% or greater.

In some embodiments, the instructions comprise a direction to administer or prescribe the one or more modified release pharmaceutical compositions in an amount sufficient to result in administration of a starting daily dose of RPPX of from about 0.1 mg/kg/day to about 1,000 mg/kg/day or from about 1 mg/kg/day to about 100 mg/kg/day. In some embodiments, the instructions comprise a direction to administer or prescribe the one or more modified release pharmaceutical compositions in an amount sufficient to result in administration of a starting daily dose of RPPX of from about 3 mg/kg/day to about 70 mg/kg/day. In some embodiments, the instructions comprise a direction to administer or prescribe the one or more modified release pharmaceutical compositions in an amount sufficient to result in administration of a starting daily dose of RPPX of from about 7 mg/kg/day to about 40 mg/kg/day. In some embodiments, the instructions comprise a direction to administer or prescribe the one or more pharmaceutical compositions in an amount sufficient to result in administration of a starting daily dose of RPPX of from about 50 mg to about 5,000 mg, from about 100 mg to about 3,000 mg, preferably from about 300 mg to about 1,500 mg, or more preferably from about 500 mg to about 1,000 mg. In some embodiments, the instructions comprise a direction to administer or prescribe the one or more modified release pharmaceutical compositions in an amount sufficient to result in administration of a starting daily dose of RPPX of from about 50 mg to about 5,000 mg, from about 100 mg to about 5,000 mg, from about 200 mg to about 5,000 mg, from about 250 mg to about 5,000 mg, from about 300 mg to about 5,000 mg, from about 400 mg to about 5,000 mg, from 450 mg to about 5,000 mg, from about 200 mg to about 3,000 mg, from about 250 mg to about 3,000 mg, from about 300 mg to about 3,000 mg, from about 400 mg to about 3,000 mg, from 450 mg to about 3,000 mg, from about 100 mg to about 1,000 mg, from about 200 mg to about 1,000 mg, from about 250 mg to about 1,000 mg, from about 300 mg to about 1,000 mg, from about 400 mg to about 1,000 mg, from about 600 mg to about 1,000 mg, or from 450 mg to about 1,000 mg. In some embodiments, the starting daily dose amount of RPPX is from about 600 mg to about 900 mg.

In some embodiments, the instructions comprise a direction to administer or prescribe the one or more modified release pharmaceutical compositions in an amount sufficient to result in administration of a starting daily dose of from about 100 mg to about 3,000 mg of RPPX to a patient. In some embodiments, instructions comprise a direction to administer or prescribe the one or more modified release pharmaceutical compositions in an amount sufficient to result in administration of a starting daily dose of from about 200 mg to about 3,000 mg of RPPX to a patient. In some embodiments, the instructions comprise a direction to administer or prescribe the one or more modified release pharmaceutical compositions in an amount sufficient to result in administration of from about 300 to about 1,500 mg of RPPX to a patient. In some embodiments, the instructions comprise a direction to administer or prescribe the one or more modified release pharmaceutical compositions in an amount sufficient to result in administration of a starting daily dose of from about 500 to about 1,000 mg of RPPX to a patient.

In some embodiments, the direction further results in administration of a non-effective dose amount of PPX. In embodiments, the non-effective dose amount of PPX is an amount that does not exceed a total dose of 1.0 mg/day. In more preferred embodiments, the non-effective dose amount of PPX is an amount that does not exceed a total dose of 0.75 mg/day, 0.5 mg/day, 0.25 mg/day, and preferably 0.125 mg/day. In some embodiments, the non-effective dose amount is less than about 0.125 mg of PPX.

In some embodiments, the direction further results in administration of a no adverse effect level (NOAEL) dose amount of PPX. In some embodiments, the no observable effective dose amount of dose of PPX may be preferably below 4.5 mg, preferably below 1.5 mg, or more preferably below 0.15 mg. In some embodiments, the no observable adverse effect level dose amount is less than about 0.05 mg per day. In another preferred embodiment, the NOAEL dose amount of PPX is an amount that does not exceed 0.0007 mg/kg per unit dose. In some embodiments, the direction further results in administration of less than about 4.5 dopaminergic activity equivalents per day. In some embodiments, the direction further results in administration of less than about 1.5 dopaminergic activity equivalents per day. In some embodiments, the direction further results in administration of less than about 0.15 dopaminergic activity equivalents per day.

In some embodiments, the instructions comprise a direction to administer or prescribe the one or more modified release pharmaceutical compositions in an amount sufficient to result in administration of a starting daily dose of from about 100 mg to 3,000 mg of RPPX to a patient; and the chiral purity for the RPPX is 99.95% or greater.

In some embodiments, the instructions comprise a direction to administer or prescribe the one or more modified release pharmaceutical compositions in an amount sufficient to result in administration of a starting daily dose of from about 200 mg to about 3,000 mg of RPPX to a patient; and the chiral purity for the RPPX is 99.95% or greater.

In some embodiments, the instructions comprise a direction to administer or prescribe the one or more modified release pharmaceutical compositions in an amount sufficient to result in administration of a starting daily dose of from about 300 mg to about 1,500 mg of RPPX to a patient; and the chiral purity for the RPPX is 99.95% or greater.

In some embodiments, the instructions comprise a direction to administer or prescribe the one or more modified release pharmaceutical compositions in an amount sufficient to result in administration of a starting daily dose of from about 500 mg to about 1,000 mg of RPPX to a patient; and the chiral purity for the RPPX is 99.95% or greater.

In some embodiments, the instructions comprise a direction to administer or prescribe the one or more modified release pharmaceutical compositions in an amount sufficient to result in administration of a starting daily dose of from about 100 mg to 3,000 mg of RPPX and less than about 0.05 mg of PPX to a patient.

In some embodiments, the instructions comprise a direction to administer or prescribe the one or more modified release pharmaceutical compositions in an amount sufficient to result in administration of a starting daily dose of from about 200 mg to 3,000 mg of RPPX and less than about 0.05 mg of PPX to a patient.

In some embodiments, the instructions comprise a direction to administer or prescribe the one or more modified release pharmaceutical compositions in an amount sufficient to result in administration of a starting daily dose of from about 300 mg to 1,500 mg of RPPX and less than about 0.05 mg of PPX to a patient.

In some embodiments, the instructions comprise a direction to administer or prescribe the one or more modified release pharmaceutical compositions in an amount sufficient to result in administration of a starting daily dose of from about 500 mg to 1,000 of RPPX and less than about 0.05 mg of PPX to a patient.

The modified release pharmaceutical or therapeutic compositions of the invention may be prepared, packaged, sold in bulk, as a single unit dose, or as multiple unit doses. The compositions may be formulated to be administered orally, ophthalmically, intravenously, intramuscularly, intra-arterially, intramedularry, intrathecally, intraventricularly, transdermally, subcutaneously, intraperitoneally, intravesicularly, intranasally, enterally, topically, sublingually, or rectally. The modified release compositions of the invention may be administered orally, preferably as a solid oral dose, and more preferably as a solid oral dose that may be a capsule or tablet. In some embodiments, the modified release compositions of the present invention may be formulated as tablets for oral administration.

The compounds of the present invention can be administered in the conventional manner by any route where they are active. Administration can be systemic, topical, or oral. For example, administration can be, but is not limited to, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, oral, buccal, or ocular routes, or intravaginally, intravesicularly, by inhalation, by depot injections, or by implants. Thus, modes of administration for the compounds of the present invention (either alone or in combination with other pharmaceuticals) can be, but are not limited to, sublingual, injectable (including short-acting, depot, implant and pellet forms injected subcutaneously or intramuscularly), or by use of vaginal creams, suppositories, pessaries, vaginal rings, rectal suppositories, intrauterine devices, and transdermal forms such as patches and creams.

The doses of the RPPX which may be administered to a patient in need thereof may range between about 0.1 mg/kg per day and about 1,000 mg/kg per day. The route of administration may include oral, sublingual, transdermal, rectal, or any accessible parenteral route. One of ordinary skill in the art will understand and appreciate the dosages and timing of the dosages to be administered to a patient in need thereof. The doses and duration of treatment may vary, and may be based on assessment by one of ordinary skill in the art based on monitoring and measuring improvements in neuronal and non-neuronal tissues. This assessment may be made based on outward physical signs of improvement, such as increased muscle control, or on internal physiological signs or markers. The doses may also depend on the condition or disease being treated, the degree of the condition or disease being treated and further on the age and weight of the patient.

Specific modes of administration will depend on the indication. The selection of the specific route of administration and the dose regimen may be adjusted or titrated by the clinician according to methods known to the clinician in order to obtain the optimal clinical response. The amount of compound to be administered may be that amount which is therapeutically effective. The dosage to be administered may depend on the characteristics of the subject being treated, e.g., the particular animal or human subject treated, age, weight, health, types of concurrent treatment, if any, and frequency of treatments, and can be easily determined by one of skill in the art (e.g., by the clinician).

A preferable route of administration of the modified release compositions of the present invention may be oral, with a more preferable route being in the form of tablets, capsules, lozenges and the like. In preferred embodiments, the compositions of the present invention may be formulated as tablets for oral administration. A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

The tablets may be uncoated or they may be coated by known techniques, optionally to delay disintegration and absorption in the gastrointestinal tract and thereby providing a sustained action over a longer period. The coating may be adapted to release the active compound in a predetermined pattern (e.g., in order to achieve a controlled release formulation) or it may be adapted not to release the active compound until after passage of the stomach (enteric coating). The coating may be a sugar coating, a film coating (e.g., based on hydroxypropyl methylcellulose, methylcellulose, methyl hydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, acrylate copolymers, polyethylene glycols and/or polyvinylpyrrolidone), or an enteric coating (e.g., based on methacrylic acid copolymer, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, shellac, and/or ethylcellulose). Furthermore, a time delay material such as, e.g., glyceryl monostearate or glyceryl distearate may be employed. The solid tablet compositions may include a coating adapted to protect the composition from unwanted chemical changes, (e.g., chemical degradation prior to the release of the active drug substance).

Modified release pharmaceutical formulations containing the compounds of the present invention and a suitable carrier may also be any number of solid dosage forms which include, but are not limited to, tablets, capsules, cachets, pellets, pills, powders and granules; topical dosage forms which include, but are not limited to, solutions, powders, fluid emulsions, fluid suspensions, semi-solids, ointments, pastes, creams, gels and jellies, and foams; and parenteral dosage forms which include, but are not limited to, solutions, suspensions, emulsions, and dry powder; comprising an effective amount of a polymer or copolymer of the present invention. It is also known in the art that the active ingredients can be contained in such formulations with pharmaceutically acceptable diluents, fillers, disintegrants, binders, lubricants, surfactants, hydrophobic vehicles, water soluble vehicles, emulsifiers, buffers, humectants, moisturizers, solubilizers, preservatives and the like. The means and methods for administration are known in the art and an artisan can refer to various pharmacologic references for guidance. For example, Modern Pharmaceutics, Banker & Rhodes, Marcel Dekker, Inc. (1979); and Goodman & Gilman's The Pharmaceutical Basis of Therapeutics, 6th Edition, MacMillan Publishing Co., New York (1980) can be consulted.

The compounds of the present invention can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. The compounds can be administered by continuous infusion over a period of about 15 minutes to about 24 hours. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The modified release compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

For oral administration, the compounds can be formulated readily by combining these compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained by adding a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients include, but are not limited to, fillers such as sugars, including, but not limited to, lactose, sucrose, mannitol, and sorbitol; cellulose preparations such as, but not limited to, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and polyvinylpyrrolidone (PVP). If desired, disintegrating agents can be added, such as, but not limited to, the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores can be provided with suitable coatings. For this purpose, concentrated sugar solutions can be used, which can optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments can be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical preparations which can be used orally include, but are not limited to, push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as, e.g., lactose, binders such as, e.g., starches, and/or lubricants such as, e.g., talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers can be added. All formulations for oral administration should be in dosages suitable for such administration.

For buccal or sublingual administration, the compositions can take the form of tablets, flash melts or lozenges formulated in any conventional manner.

For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount, Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The compounds of the present invention can also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds of the present invention can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Depot injections can be administered at about 1 to about 6 months or longer intervals. Thus, for example, the compounds can be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

In transdermal administration, the compounds of the present invention, for example, can be applied to a plaster, or can be applied by transdermal, therapeutic systems that are consequently supplied to the organism.

Pharmaceutical and therapeutic compositions of the compounds also can comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as, e.g., polyethylene glycols.

The compounds of the present invention can also be administered in combination with other active ingredients, such as, for example, adjuvants, protease inhibitors, or other compatible drugs or compounds where such combination is seen to be desirable or advantageous in achieving the desired effects of the methods described herein.

Preparation of (R)- and PPX

Processes for the preparation of pramipexole are described in U.S. Pat. Nos. 4,843,086 and 4,886,812 to Griss et al., each of which is incorporated by reference in its entirety. The RPPX of the present invention may be synthesized and/or purified by methods disclosed in the copending U.S. Provisional Application No. 60/894,829 entitled “Methods of Synthesizing and Purifying R(+) and (S)-pramipexole”, filed on Mar. 14, 2007, and U.S. Provisional Application No. 60/894,814 entitled “Methods of Enantiomerically Purifying Chiral Compounds”, filed on Mar. 14, 2007 and, ______filed simultaneously herewith, which are incorporated herein by reference in their entireties. Specifically, preparations which are chirally pure for the R(+) enantiomer may be produced using a bi-molecular nucleophilic substitution (S_(N)2) reaction. A diamine, 2,6 diamino-4,5,6,7-tetrahydro-benzothiazole, is reacted with a propyl sulfonate or a propyl halide in polar solvents to generate an insoluble pramipexole salt in a one pot synthesis scheme. The 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole salt reaction product displays a high chemical purity and an increased optical purity over the reactants, which may be due to limited solubility of the 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole salt in the polar solvents of the reaction mixture. Purification of the final 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole synthesis product from the reaction mixture thus involves simple trituration and washing of the precipitated 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole salt in a volatile solvent such as an alcohol or heptane, followed by vacuum drying.

In some embodiments, the RPPX is prepared by dissolving a diamine of formula 2,6 diamino-4,5,6,7-tetrahydro-benzothiazole in an organic solvent, reacting the diamine with a propyl sulfonate or a propyl halide under conditions sufficient to generate and precipitate the 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole salt, and recovering the 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole salt. In a preferred embodiment, the propyl sulfonate may be propyl tosylate. In a further embodiment, the propyl halide may be propyl bromide. The 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole salt reaction product of this process displays a high chemical purity and an increased optical purity over the reactants. Without wishing to be bound by theory, the increased optical purity may be due to limited solubility of the 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole salt reaction product in the polar solvents of the reaction mixture. Purification of the final 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole reaction product from the reaction mixture thus involves simple trituration and washing of the precipitated 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole salt in a volatile solvent such as an alcohol or heptane, followed by vacuum drying.

In embodiments of the process, the diamine may be an R(+) diamine, or a mixture of the R(+) and an S diamine. The chemical purity of the final 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole salt may be at least about 97% or greater, preferably 98% or greater, more preferably 99% or greater. The R(+) enantiomers of the 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole salt generated using this process are generated from starting diamines which may be at least 55% optically pure, preferably 70% optically pure, and more preferably greater than 90% optically pure. The final 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole product may be enriched to 99.6% optical purity or greater, 99.7% optical purity or greater, preferably 99.8% optically purity or greater, and more preferably 99.9% optical purity or greater, 99.95% optical purity or greater, 99.99% optical purity or greater. In some embodiments, the optical purity may be 100%.

In embodiments of the process, the organic solvent may be a polar aprotic solvent such as tetrahydrofuran, dimethylformamide, dimethyl sulfoxide dimethylacetamide, or hexamethylphosphoric triamide. The organic solvent may also be a low molecular weight alcohol such as ethanol, 1-propanol, or n-butanol. Further, the organic solvent may be any combination of the polar aprotic solvents and low molecular weight alcohols. The organic solvent may have a water content of from about 0 to about 10 volume percent. Preferably, the solvents used in the practice of this invention are standard ACS grade solvents. Further, the propyl sulfonate or a propyl halide may be added at about 1.0 to about 2.0 molar equivalents of the diamine.

In further embodiments of the process, the conditions sufficient to generate and precipitate the 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole salt may comprise heating the dissolved diamine at an elevated temperature, adding the propyl sulfonate or propyl halide which may be dissolved in di-isoproplyethylamine and an organic solvent to form a mixture, and stirring the mixture for about 4 hours. Alternatively, the di-isoproplyethylamine may be added to the reaction with the diamine, and the propyl sulfonate or propyl halide may be dissolved in an organic solvent to form a mixture, which may be added to the reaction with stirring over about 4 hours.

In this embodiment, the elevated temperature of the reaction may be below the boiling temperature of the reaction, specifically, below the boiling temperature of the organic solvent(s) of the reaction mixture. The elevated temperature may be lower than about 125° C., preferably lower than about 100° C., and more preferably about 95° C. or lower. The times necessary for the reaction may vary with the identities of the reactants, the solvent system and with the chosen temperature.

In an alternative embodiment, the conditions sufficient to generate and precipitate the 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole salt may comprise using dimethylformamide as the organic solvent, heating the dissolved diamine at an elevated temperature, adding the propyl sulfonate or propyl halide which is dissolved in dimethylformamide to form a mixture, and stirring the mixture for about 4 hours. The elevated temperature of the reaction may be below the boiling temperature of the reaction, specifically, below the boiling temperature of the organic solvent(s) of the reaction mixture. The elevated temperature may be lower than about 125° C., preferably lower than about 100° C., and more preferably about 75° C. or lower. The times necessary for the reaction may vary with the identities of the reactants, the solvent system and with the chosen temperature.

In a preferred alternative embodiment, the conditions sufficient to generate and precipitate the 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole salt comprise using dimethylformamide as the organic solvent and heating the dissolved diamine at an elevated temperature. A mixture of propyl sulfonate or propyl halide, at preferably 1.25 molar equivalents, dissolved in dimethylformamide, preferably 10 volumes, and di-isoproplyethylamine, preferably 1.25 molar equivalents, is added slowly to the heated diamine with stirring over a period of about 4 hours. Alternatively, the di-isoproplyethylamine may be added to the reaction with the diamine, and the propyl sulfonate or propyl halide may be dissolved in dimethylformamide to form a mixture, which may be added to the reaction with stirring for about 4 hours. The elevated temperature of the reaction may be below the boiling temperature of the reaction, specifically, below the boiling temperature of the organic solvent(s) of the reaction mixture. The elevated temperature may be lower than about 125° C., preferably lower than about 100° C., and more preferably about 65° C. or lower. The times necessary for the reaction may vary with the identities of the reactants, the solvent system and with the chosen temperature.

Embodiments of the process further comprise cooling the reaction to a temperature of about room temperature, about 25° C., and stirring the reaction for about 2 hours. The process may further involve filtering the reaction to isolate a solid precipitate, washing the precipitate with an alcohol, and drying the precipitate under vacuum. The 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole salt reaction product of this process may display an increased optical purity over the reactants.

Alternatively, the 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole sulfonate or halide salt can be reacted with concentrated HCl in an organic solvent, such as an alcohol, at a temperature of from about 0 to about 5° C. An organic solvent, such as methyl tert-butyl ether (MTBE), may be added, and the reaction may be stirred for an additional hour. The RPPX dihydrochloride product may be recovered from the reaction mixture by filtering, washing with an alcohol and vacuum drying.

In an embodiment of the process, referred to as condition A in Table 7 and in the examples, the reaction condition which may be sufficient to generate the 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole product may include heating the dissolved diamine of formula II to an elevated temperature with continuous stirring. The elevated temperature is preferably less than the melting point of the chosen organic solvent, lower than about 125° C., preferably lower than about 110° C., and more preferably about 95° C. A solution of propyl sulfonate or propyl halide, which is dissolved in di-isoproplyethylamine and an organic solvent to form a mixture, is added slowly over a period of several hours. This reaction mixture may then be stirred at temperature for an additional period of time such as, for example, about 4 hours. The times necessary for the reaction may vary with the identities of the reactants and solvent system, and with the chosen temperature, and would be understood by one of skill in the art.

In an alternate embodiment, the di-isoproplyethylamine may be added to the reaction with the diamine, and the propyl sulfonate or propyl halide may be dissolved in an organic solvent to form a mixture, which may be added to the reaction with stirring over a period of several hours. This reaction mixture may then be stirred at temperature for an additional period of time such as, for example, at least 4 hours. The time necessary for the reaction to run to completion may vary with the identities of the reactants and solvent system, and with the chosen temperature, and would be understood by one of skill in the art.

In an alternative embodiment of the process, referred to as condition B in Table 7, the reaction conditions which are sufficient to generate the 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole product may include using dimethylformamide as the organic solvent, and heating the dissolved diamine of formula II to an elevated temperature with continuous stirring. The elevated temperature is preferably less than the melting point of the chosen organic solvent, lower than about 125° C., preferably lower than about 100° C., and more preferably about 75° C. A solution of propyl sulfonate or propyl halide, which is dissolved in dimethylformamide, may be added slowly over a period of several hours. This reaction mixture may then be stirred at temperature for an additional period of time such as, for example, about 4 hours. The time necessary for the reaction to run to completion may vary with the identities of the reactants and solvent system, and with the chosen temperature, and would be understood by one of skill in the art.

In a preferred alternative embodiment of the process, referred to as condition C in Table 7, the reaction includes using dimethylformamide as the organic solvent for dissolution of the diamine. The diamine of formula II may then be heated to an elevated temperature with continuous stirring. The elevated temperature is preferably less than the melting point of the chosen organic solvent, lower than about 125° C., preferably lower than about 100° C., and more preferably about 65° C. A solution of propyl sulfonate or propyl halide, preferably about 1.25 molar equivalents, may be dissolved in dimethylformamide, preferably about 10 volumes, and di-isoproplyethylamine, preferably about 1.25 molar equivalents, to form a mixture. This mixture may be added slowly over a period of several hours to the heated diamine. This reaction mixture may then be stirred at temperature for an additional period of time such as, for example, about 4 hours. Alternatively, the di-isoproplyethylamine may be added to the reaction with the diamine, and the propyl sulfonate or propyl halide may be dissolved in dimethylformamide to form a mixture, which may be added to the reaction with stirring over a period of several hours. This reaction mixture may then be stirred at temperature for an additional period of time such as, for example, about 4 hours. The time necessary for the reaction to run to completion may vary with the identities of the reactants and solvent system, and with the chosen temperature, and would be understood by one of skill in the art.

Purification of the final 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole product may include cooling the reactions disclosed above to a temperature of about 25° C., and stirring the reactions for a period of time such as, for example, about 2 hours. The purification may further include filtering the reaction to isolate a solid precipitate, washing the precipitate with an alcohol, and drying the precipitate under vacuum. The final products of the reaction may be analyzed by high pressure liquid chromatography (HPLC) for chemical and chiral purity.

Further, ¹H NMR and ¹³C NMR may be used to confirm the structure of the product 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole. Results of example syntheses using each of the several conditions which are embodiments of the present disclosure are listed in Table 7. Several example syntheses of 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole using conditions A and C of the present disclosure are detailed in Examples 5-7.

The sulfonate or halide salts of 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may be converted to an HCl salt using a concentrated solution of HCl in ethanol. A p-TSA 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole salt may be re-dissolved in an alcohol, such as ethanol, and the mixture may be cooled to between about 0 and about 5° C. with continuous stirring. A concentrated HCl may then be added, followed by a solvent such as methyl tert-butyl ether (MTBE), and the mixture may be stirred for an hour at between about 0 and about 5° C. The reaction mixture may then be filtered, washed with an MTBE/alcohol solution, and dried under vacuum. The final product is 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole dihydrochloride. A detailed example of this synthesis may be found in Example 8.

An alternate method for conversion of the sulfonate or halide salts of 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole to an HCl salt involves the use of a concentrated solution of HCl and isopropyl acetate (IPAC). A sulfonate or halide salt of 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may be taken up in IPAC and cooled to 15° C. HCl (gas) may be bubbled into the slurry for about 1 hour, after which the mixture may be filtered, washed with IPAC and dried under vacuum at room temperature to afford a 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole dihydrochloride salt. A detailed example of this synthesis may be found in Example 9.

The sulfonate or halide salts of 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may alternatively be converted to the free base form of 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole. A p-TSA 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole salt may be dissolved in dichloromethane (DCM) and water. The resulting solution may then by brought to a pH of about 11-12 using NaOH. Two phases may be generated, and the aqueous phase may be extracted with DCM, dried over magnesium sulfate (MgSO₄), filtered over Celite® and concentrated. The concentrated residue may be re-dissolved in MTBE and stirred as a slurry for several hours. The solids may then be filtered, washed with MTBE, and dried under vacuum at a temperature of about 35° C. The final product is 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole free base. A detailed example of this synthesis may be found in Example 10.

Alternatively, the sulfonate or halide salts of 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may alternatively be converted to the free base form of 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole by a second process. In this second process, the p-TSA salt of 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole is dissolved in water and cooled to a temperature of about 10° C. This slurry is basified by addition of NaOH, diluted with brine, and extracted several times in DCM. The combined organic phases are then washed with brine, dried over MgSO₄, filtered and concentrated to dryness. A detailed example of this synthesis may be found in Example 11.

The free base form of 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may be converted to 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole dihydrochloride by bubbling HCl gas into a cooled solution of the 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole free base in IPAC. Alternatively, the free base form of 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may be converted to 2-amino-4,56,7-tetrahydro-6-(propylamino)benzothiazole dihydrochloride by mixing with concentrated HCl at room temperature overnight. Detailed examples of the aforementioned synthesis schemes may be found in Examples 12 and 13, respectively.

Alternatively, the free base form of 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may be converted to 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole fumarate by the addition of 2 molar equivalents of fumaric acid.

TABLE 7 Experiments for S_(N)2 preparation of 2-amino-4,5,6,7-tetrahydro-6- (propylamino)benzothiazole pTSA salt Condition Isomer Batch Size Results A R(+) 45 grams Yield = 53.2 grams (52%) Chemical Purity = 98.2% AUC by HPLC Chiral Purity = >99.5% AUC by HPLC A S(−) 5 grams Yield = 4.99 grams (44.2%) Chemical Purity = 98.0% AUC by HPLC Chiral Purity = >99.6% AUC by HPLC A Racemic 5 gram Yield = 5.12 grams (45%) Chemical Purity = 97.1% AUC by HPLC Chiral Purity = 1:1 R(+):S(−) by HPLC B R(+) 5 gram Yield = 4.6 grams (40%) Chemical Purity = 94.9% AUC by HPLC Chiral Purity = 99.6% AUC by HPLC B S(−) 10 gram Yield = 9.81 grams (43.3%) Chemical Purity = 94.9% AUC by HPLC Chiral Purity = 99.7% AUC by HPLC B Racemic 5 gram Yield = 2.9 grams (25.6%) Chemical Purity = 98.3% AUC by HPLC Chiral Purity = 1:1 R(+):S(−) by HPLC C R(+) 250 gram Yield = 317.6 grams (56%) Chemical Purity = 99.4% AUC by HPLC Chiral Purity = 99.8% AUC by HPLC C S(−) 20 gram Yield = 25.41 grams (56%) Chemical Purity = 99.4% AUC by HPLC Chiral Purity = 99.7% AUC by HPLC C Racemic 5 gram Yield = 6.02 grams (53.1%) Chemical Purity = 99.2% AUC by HPLC Chiral Purity = 1:1 R(+):S(−) by HPLC E* R(+) 25 gram Yield = 47% Chiral Purity = 99.8% AUC by HPLC E* S(−) 25 gram Yield = 47% Chiral Purity = 99.8% AUC by HPLC *Condition E is the same as Condition C, except that the recovery step does not incorporate dilution in MTBE. The MTBE increases the recovery (yield) from the synthesis reaction, but may reduce the overall chiral purity. Condition E is explained in more detail in Table 9.

The alternative process for preparing an enantiomerically pure material from a mixture of RPPX and PPX involves using acid addition and trituration (precipitation) of an enantiomerically pure 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole based on insolubility of the enantiomers (R(+) and S(−)) in the resulting achiral salt solution. In embodiments of this process, enantiomerically pure 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole is triturated from an acid addition solution based on the insolubility of the enantiomers in the resulting achiral salt reagents. This embodiment, a process for preparing an enantiomerically pure 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole, comprises dissolving an enantiomerically enriched 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole in an organic solvent at an elevated temperature, adding a selected acid, cooling the reaction to room temperature, stirring the cooled reaction at room temperature for an extended time and recovering enantiomerically pure RPPX. In a preferred embodiment, the selected acid may be added at from about 1 molar equivalent to about 2 molar equivalents of the enantiomerically enriched 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole.

In an embodiment of the process, the selected acid is p-toluenesulfonic acid (p-TSA) and the organic solvent is ethanol. In another embodiment of the process, the elevated temperature may be from about 65° C. to about 85° C. and the cooling occurs at a rate of about 25° C. per hour. The elevated temperature may also be a temperature lower than 125° C., preferably lower than 100° C. and more preferably about 75° C. The times necessary for the reaction may vary with the identities of the reactants, the solvent system and with the chosen temperature, and may be easily appreciated by one of skill in the art. In yet another embodiment of the process, recovering enantiomerically pure 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole comprises cooling the reaction to a temperature of about 25° C. and stirring the reaction for at least about 2 hours. The recovery may further comprise filtering the reaction to isolate a solid precipitate, washing the precipitate with an alcohol and drying the precipitate under vacuum.

In various embodiments of the process, the organic solvent may include, but is not limited to, acetonitrile, acetone, ethanol, ethyl acetate, methyl tert-butyl ether, methyl ethyl ketone, isopropyl acetate and isopropyl alcohol. In a preferred embodiment, the organic solvent is ethanol. The acid may include, but is not limited to, halogenic acids such as, for example, hydrobromic, hydrochloric, hydrofluoric and hydroiodic acid; inorganic acids such as, for example, nitric, perchloric, sulfuric and phosphoric acid; organic acids such as, for example, sulfonic acids (methanesulfonic, trifluoromethan sulfonic, ethanesulfonic, benzenesulfonic or p-toluenesulfonic), acetic, malic, fumaric, succinic, citric, benzoic, gluconic, lactic, mandelic, mucic, pamoic, pantothenic, oxalic and maleic acid; and aminoacids such as aspartic or glutamic acid. The acid may be a mono- or di-acid, such as a di-hydrohalogenic, di-sulfuric, di-phosphoric or di-organic acid. In all cases, the acid is used as an achiral reagent which is not selected on the basis of any expected or known preference for interaction with or precipitation of a specific optical isomer of the products of this disclosure. In a preferred embodiment, the selected acid is p-toluenesulfonic acid.

In embodiments of the process, the final chiral purity for an R(+) enantiomer of the 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole salt may be greater than 99% when the starting mixture contains 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole which is at least 55% optically pure for the R(+) enantiomer, preferably 80% optically pure for the R(+) enantiomer, preferably 85% optically pure for the R(+) enantiomer, more preferably 90% optically pure for the R(+) enantiomer and most preferably 95% optically pure for the R(+) enantiomer. The final chiral purity for an S(−) enantiomer of the 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole salt may be greater than 99% when the starting mixture contains 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole which is at least 55% optically pure for the S(−) enantiomer, preferably 80% optically pure for the S(−) enantiomer, preferably 85% optically pure for the S(−) enantiomer, more preferably 90% optically pure for the S(−) enantiomer and most preferably 95% optically pure for the S(−) enantiomer. The chiral purity of the final 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole salt may preferably be 99.6% or greater, 99.7% or greater, preferably 99.8% or greater, and more preferably 99.9% or greater. In some embodiments, the chiral purity of the final 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole salt may be 100%.

In embodiments, after the enantiomerically enriched 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole is dissolved in an organic solvent at an elevated temperature and the acid is added, the reaction may be cooled to room temperature at a rate of about 25° C./hour. The enantiomerically pure 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may then be recovered from the reaction solution by stirring the reaction for at least about 2 hours, filtering the reaction to isolate a solid precipitate, washing the precipitate with an alcohol and drying the precipitate under vacuum. The rates of cooling and the time required for the additional stirring may vary with the chosen organic solvent and acid, and may be easily appreciated by one skilled in the art. Additionally, the reaction volumes may dictate the degree of optical purification and the overall yield of the final 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole product. These volumes would be understood and appreciated by one of skill in the art. Examples of specific times, temperatures and volumes which enable the practice of this invention are given in the Examples.

In embodiments, the chiral purity of the 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole salt product for the R(+) enantiomer may be greater than 99% when the chiral purity of the starting 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole mixture for the R(+) enantiomer is greater than 55%, preferably greater than 70%, or more preferably greater than 90%. The chiral purity of the final 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole salt may be 99.6% or greater, 99.7% or greater, preferably 99.8% or greater, and more preferably 99.9% or greater, more preferably 99.95% or greater, even more preferably 99.99% or greater. In some embodiments, the chiral purity of the final 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole salt may be 100%.

Chirally pure 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole also may be prepared by the process of trituration of a single enantiomer of 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole from a mixture of RPPX and PPX by acid addition, based on insolubility of the enantiomers in the resulting achiral salt solution. The process comprises dissolving an enantiomerically enriched 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole in an organic solvent at an elevated temperature, adding from about 1.05 molar equivalents to about 2.05 molar equivalents of a selected acid, cooling the reaction to room temperature, stirring the cooled reaction at room temperature for an extended time and recovering enantiomerically pure 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole.

In embodiments, the elevated temperature of the reaction may be below the boiling temperature of the reaction, specifically, below the boiling temperature of the organic solvent(s) of the reaction mixture. The elevated temperature may be lower than about 125° C., more preferably lower than about 100° C., and more preferably about 75° C. The times necessary for the reaction may vary with the identities of the reactants, the solvent system and with the chosen temperature, and would be appreciated by one of skill in the art.

In embodiments, the organic solvent may include, but is not limited to, acetonitrile, acetone, ethanol, ethyl acetate, methyl tert-butyl ether, methyl ethyl ketone, isopropyl acetate and isopropyl alcohol. In a preferred embodiment, the organic solvent is ethanol. In this embodiment, the acid may include, but is not limited to, halogenic acids such as, for example, hydrobromic, hydrochloric, hydrofluoric and hydroiodic acid; inorganic acids such as, for example, nitric, perchloric, sulfuric and phosphoric acid; organic acids such as, for example, sulfonic acids (methanesulfonic, trifluoromethan sulfonic, ethanesulfonic, benzenesulfonic or p-toluenesulfonic), acetic, malic, fumaric, succinic, citric, benzoic, gluconic, lactic, mandelic, mucic, pamoic, pantothenic, oxalic and maleic acid; and aminoacids such as aspartic or glutamic acid. The acid may be a mono- or di-acid, such as a di-hydrohalogenic, di-sulfuric, di-phosphoric or di-organic acid. In all cases, the acid is used as an achiral reagent which is not selected on the basis of any expected or known preference for interaction with or precipitation of a specific optical isomer of the products of this disclosure. In a preferred embodiment, the selected acid is p-toluenesulfonic acid.

In additional embodiments, after the enantiomerically enriched 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole is dissolved in an organic solvent at an elevated temperature and the acid is added, the reaction may be cooled to room temperature at a rate of about 25° C./hour. The chirally pure 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may then be recovered from the reaction solution by stirring the reaction for at least about 2 hours, filtering the reaction to isolate a solid precipitate, washing the precipitate with an alcohol and drying the precipitate under vacuum. The rates of cooling and the time required for the additional stirring may vary with the chosen organic solvent and acid, and would be appreciated by one skilled in the art. Additionally, the reaction volumes may dictate the degree of optical purification and the overall yield of the final 2-amino-4,5,6,7-tetrahydro-6-(propylamino benzothiazole product. These volumes would be understood and appreciated by one of skill in the art. Examples of specific times, temperatures and volumes which enable the practice of this invention are given in the Examples.

In embodiments, the chiral purity for the R(+) enantiomer of the recovered 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole salt may be greater than 99% when the starting 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole material has a chiral purity for the R(+) enantiomer of greater than 55%, preferably greater than 70%, or more preferably greater than 90%. The chiral purity of the final 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole salt for the R(+) enantiomer may be 99.6% or greater, 99.7% or greater, preferably 99.8% or greater, and more preferably 99.9% or greater, more preferably 99.95% or greater, even more preferably 99.99% or greater. In a most preferred embodiment, the chiral purity of the final 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole salt for the R(+) enantiomer may be 100%.

The process may include dissolving an enantiomerically enriched 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole in an organic solvent at an elevated temperature, adding from about 1.05 equivalents to about 2.05 equivalents of a selected acid, cooling the reaction to room temperature, stirring the cooled reaction at room temperature for an extended period of time and recovering enantiomerically pure 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole of formula I.

In an embodiment of the process, the selected acid is p-toluenesulfonic acid (p-TSA) and the organic solvent is ethanol. In another embodiment of the process, the elevated temperature may be from about 65° C. to about 85° C. and the cooling occurs at a rate of about 25° C. per hour. The elevated temperature may also be a temperature lower than 125° C., preferably lower than 100° C., and more preferably about 75° C. The times necessary for the reaction may vary with the identities of the reactants, the solvent system and with the chosen temperature, and may be easily appreciated by one of skill in the art. In yet another embodiment of the process, recovering enantiomerically pure 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole comprises cooling the reaction to a temperature of about 25° C. and stirring the reaction for at least about 2 hours. The recovery may further comprise filtering the reaction to isolate a solid precipitate, washing the precipitate with an alcohol and drying the precipitate under vacuum.

In various embodiments of the process, the organic solvent may include, but is not limited to, acetonitrile, acetone, ethanol, ethyl acetate, methyl tert-butyl ether, methyl ethyl ketone, isopropyl acetate and isopropyl alcohol. In a preferred embodiment, the organic solvent is ethanol. The acid may include, but is not limited to, halogenic acids such as, for example, hydrobromic, hydrochloric, hydrofluoric and hydroiodic acid; inorganic acids such as, for example, nitric, perchloric, sulfuric and phosphoric acid; organic acids such as, for example, sulfonic acids (methanesulfonic, trifluoromethan sulfonic, ethanesulfonic, benzenesulfonic or p-toluenesulfonic), acetic, malic, fumaric, succinic, citric, benzoic, gluconic, lactic, mandelic, mucic, pamoic, pantothenic, oxalic and maleic acid; and aminoacids such as aspartic or glutamic acid. The acid may be a mono- or di-acid, such as a di-hydrohalogenic, di-sulfuric, di-phosphoric or di-organic acid. In all cases, the acid is used as an achiral reagent which is not selected on the basis of any expected or known preference for interaction with or precipitation of a specific optical isomer of the products of this disclosure. In a preferred embodiment, the selected acid is p-toluenesulfonic acid.

In embodiments of the process, the final chiral purity for an R(+) enantiomer of the 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole salt may be greater than 99% when the starting mixture contains 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole which is at least 55% optically pure for the R(+) enantiomer, preferably 80% optically pure for the R(+) enantiomer, preferably 85% optically pure for the R(+) enantiomer, more preferably 90% optically pure for the R(+) enantiomer and most preferably 95% optically pure for the R(+) enantiomer. The final chiral purity for an S(−) enantiomer of the 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole salt may be greater than 99% when the starting mixture contains 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole which is at least 55% optically pure for the S(−) enantiomer, preferably 80% optically pure for the S(−) enantiomer, preferably 85% optically pure for the S(−) enantiomer, more preferably 90% optically pure for the S(−) enantiomer and most preferably 95% optically pure for the S(−) enantiomer. The chiral purity of the final 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole salt may preferably be 99.6% or greater, 99.7% or greater, preferably 99.8% or greater, and more preferably 99.9% or greater. In a more preferred embodiment, the chiral purity of the final 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole salt may be 100%.

Results of example purifications using each of the several conditions which are embodiments of the present disclosure are listed in Table 8.

TABLE 8 Experiments for preparation of the R(+) enantiomer of 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole (RPPX) Acid Solvent Batch Size Results p-TSA ethanol 298.7 mg Yield = 489.5 mg (90.3%) Start Chiral Purity = 91% AUC R(+) by HPLC Final Chiral Purity = 100% AUC by HPLC MSA acetonitrile 300.0 mg Yield = 431.8 mg (98.9%) Start Chiral Purity = 91% AUC R(+) by HPLC Final Chiral Purity = 99.23% AUC by HPLC fumaric acetonitrile 301.0 mg Yield = 532 mg (84.2%) (hot ethanol) Start Chiral Purity = 91% AUC R(+) by HPLC Final Chiral Purity = 99.26% AUC by HPLC phosphoric acetonitrile 299.4 mg Yield = 592 mg (~100%) Start Chiral Purity = 91% AUC R(+) by HPLC Final Chiral Purity = 100% AUC by HPLC

The chemical and chiral purity of the preparations of RPPX may be verified with at least HPLC, ¹³C-NMR, ¹H-NMR and FTIR. In preferred embodiments, the RPPX may be synthesized by the method described above, which yields enantiomerically pure material. Alternatively, the RPPX may be purified from mixtures of RPPX and PPX using a purification scheme which is disclosed in copending U.S. Provisional Application No. 60/894,829 entitled “Methods of Synthesizing and Purifying R(+) and (S)-pramipexole”, filed on Mar. 14, 2007, and U.S. Provisional Application No. 60/894,814 entitled “Methods of Enantiomerically Purifying Chiral Compounds”, filed on Mar. 14, 2007, which are incorporated herein by reference in their entireties.

By way of explanation, and not wishing to be bound by theory, the solubility of RPPX and PPX may be the same in the trituration step of the synthesis and purification processes. As example, if a synthesis process is carried out with 90 grams of the R(+) diamine and 10 grams of the S(−)diamine, and the solubility of the final 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole product is 10 grams for either enantiomer, then 80 grams of the RPPX product and 0 grams of the PPX product would precipitate (assuming a 100% chemical conversion from the diamine and no change in molecular weight in going to the 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole product). That is, 10 grams of each enantiomer of 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may be expected to go into solution. This would lead to a 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole product with a 100% chiral purity for the R(+) enantiomer. The opposite ratio of starting materials for the synthesis process (90 grams of the S(−) diamine and 10 grams of the R(+) diamine) may generate a reaction product of 90 grams of the PPX and 10 grams of the RPPX. From this reaction product mixture, 80 grams of the S(−) enantiomer and 0 grams of the R(+) enantiomer of 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole would be expected to precipitate, leading to a 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole product with a 100% chiral purity for the S(−) enantiomer. In this thought experiment, one can imagine that the volumes which are used for a reaction may have a large potential effect on the final yield and chiral purity. That is, too large a volume will reduce the yield as more of the 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole enantiomer products will go into solution (but increase the chiral purity) and too small a volume will increase the yield as less of the 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole products will go into solution (but reduce the chiral purity).

To better define the actual limits of the reaction volumes and optical purities attainable using methods of the disclosure, various ratios of chiral purity for the starting diamine material were tested. As shown in Table 9, the synthesis and purification process was tested using the following ratios of the starting R(+) and S(−) diamine: 80:20, 20:80, 85:15, 15:85, 90:10, 10:90, 95:5 and 5:95. Additionally, three specific reaction conditions were tested which varied either the reaction volume or a post reaction recovery step. These trials demonstrated that the enantiomers of 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole are equally insoluble (or soluble) in the organic solvents utilized in the various embodiments of the synthesis processes disclosed herein.

TABLE 9 Experiments for S_(N)2 preparation of pure enantiomers of 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole Ratio of Condition C Condition D Condition E starting diamines (yield/chiral (yield/chiral (yield/chiral R(+):S(−) purity) purity) purity) 80:20 — 29%/99%   34%/98.2% 20:80 — 30%/99.4% 35%/95.7% 85:15 43%/86.8% 36%/99.8% 39%/99.9% 15:85 52%/88.9% 27%/99.6% 37%/99.9% 90:10 47%/95.9% — — 10:90 58%/93.6% — — 95:5  50%/99.6% — —  5:95 47%/99.6% — — Condition C: The reaction is performed in 10 volumes of DMF and 1.25 equivalents of propyl tosylate at 65-67° C. The reaction is then cooled to room temperature and diluted with 8 volumes of MTBE. Condition D: The reaction is performed in 18 volumes of DMF and 1.25 equivalents of propyl tosylate at 65-67° C. The reaction is then cooled to room temperature and diluted with 8 volumes of MTBE. Condition E: The reaction is performed in 10 volumes of DMF and 1.25 equivalents of propyl tosylate at 65-67° C. The reaction is then cooled to room temperature with no dilution in MTBE.

The data in Table 9 demonstrate that both enantiomers of 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole have similar, if not the same, solubility. Further, the data show that the synthesis is equally efficient for either enantiomer of 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole. These data also demonstrate that the enantiomers behave independently of one another, in that the solubility of one enantiomer appears to be unaffected by the concentration in solution of the other. For example, the various synthesis reactions carried out using condition C all have chemical yields of about 50%, independent of the percentage of predominant diamine enantiomer of the starting material. When the volume of the organic solvent used in the synthesis reaction is increased, the chemical yield is reduced, but the chiral yield is increased. This is apparent by comparison of the reaction carried out in conditions C and P, where an 85:15 ratio of R(+):S(−) diamine produced a 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole product having an 86.8% chiral purity for the R(+) enantiomer when the reaction used 10 volumes of the organic solvent and a 99.8% chiral purity for the R(+) enantiomer when the reaction used 18 volumes of the organic solvent. Note also that the chemical yield was reduced in the reaction using a larger volume of organic solvent (43% yield in condition C and 36% yield in condition D).

In Table 9, condition E is the same as condition C, except that the recovery step does not incorporate dilution in MTBE. The MTBE is observed to increase 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole recovery (yield) from the synthesis reaction, but may reduce the overall chiral purity. This is born out by a comparison of the results for trials carried out in an 85:15 ratio of R(+):S(−) diamine, which produced a 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole product having a 86.8% chiral purity for the R(+) enantiomer when the reaction included the MTBE organic solvent and a 99.90/0 chiral purity for the R(+) enantiomer when the reaction did not include the MTBE organic solvent. The chemical yield was reduced by exclusion of the MTBE dilution in the recovery step; a 43% yield in condition C as opposed to a 39% yield in condition E.

The chirally pure RPPX prepared by any of the above methods may be converted to a pharmaceutically acceptable salt of RPPX. For example, RPPX dihydrochloride is a preferred pharmaceutical salt due its high water solubility. RPPX dihydrochloride may be prepared from other salts of RPPX in a one step method comprising reacting the RPPX, or RPPX salt, with concentrated HCl in an organic solvent, such as an alcohol, at a reduced temperature. In some embodiments, the reduced temperature is a temperature of from about 0° C. to about 5° C. An organic solvent, such as methyl tert-butyl ether, may be added, and the reaction may be stirred for an additional hour. The RPPX dihydrochloride product may be recovered from the reaction mixture by filtering, washing with an alcohol and vacuum drying.

Each of the methods disclosed herein for the manufacture and purification of RPPX or a pharmaceutically acceptable salt thereof may be scalable to provide industrial scale quantities and yields, supplying products with both high chemical and chiral purity. In some embodiments, the enantiomerically pure RPPX may be manufactured in large batch quantities as may be required to meet the needs of a large scale pharmaceutical use.

Various aspects of the present invention will be illustrated with reference to the following non-limiting examples.

EXAMPLES Example 1

Measurement of the dopamine receptor affinities for the R(+) and S(−) enantiomers of 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole.

The S(−) enantiomer of 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole has historically been characterized as a high affinity dopamine receptor ligand at the D₂ (both the S and L isoforms), 93 and D₄ receptors, although the highest affinity is seen for the D₃ receptor subtype. The dopamine receptor ligand affinity of PPX and of RPPX from journal publications has been tabulated (data are reproduced in Table 10). Although the conditions under which each study or experiment was carried out are slightly different, and different radio-ligands were used, the data show comparable affinities for the various dopamine receptors. Studies we conducted on the dopamine receptor affinities of the S(−) and the R(+) enantiomers of 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole are also shown in Table 10. These data demonstrate an unexpectedly large difference in the affinities of the two enantiomers of 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole for all dopamine receptors. Table 10 shows that; instead of the expected 10-20 fold difference in binding affinity for 92 receptor affinity, and 50-fold difference in binding affinity for D3 receptor affinity as derived from the literature, the values we found were typically 10-fold higher (290- and 649-fold, respectively) (Table 10).

TABLE 10 Comparative binding affinity data for 2-amino-4,5,6,7- tetrahydro-6-(propylamino)benzothiazole enantiomers Receptor Source S(−) R(+) Ratio S:R D₂ binding, IC₅₀ (nM) Lit.* 4,700 43,000 9 D₂ binding, IC₅₀ (nM) Lit.** 402 8,330 21 D₂ binding, IC₅₀ (nM) Actual*** 6.2 1,800 290 D₃ binding, IC₅₀ (nM) Lit.** 4.2 211 50 D₃ binding, IC₅₀ (nM) Actual*** 0.94 610 649 *Schneider, C. S.; Mierau, J., “Dopamine Autoreceptor Agonists: Resolution and Pharmacological Activity of 2,6-Diaminotetrahydrobenzothiazole and an Aminothiazole Analogue of Apomorphine”, (1987). J. Med. Chem. 30: 494-498 **Wong, S. K.-F.; Shrikhande, A. V., S. K.-F. Wong, “Activation of Extracellular Signal-Regulated Kinase by Dopamine D2 and D3 Receptors”,. (2003) Society for Neuroscience Abstracts ***Data from current study

The RPPX and PPX were supplied as dry powder to our contract research partner Cerep by the manufacturer AMRI. Solutions of RPPX and PPX were prepared from stock solutions in DMSO. Eight concentrations of RPPX or PPX (0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 mM, 1 mM and 100 mM) were used to displace standard reference radiolabeled dopamine agonists. These concentrations were tested in cell lines expressing select human cloned dopamine receptors (D_(2S), D₃). Previous work in the literature and our data demonstrated no significant interaction with D1 and D5 dopamine receptors. Group results for the interaction of RPPX or PPX with each receptor are expressed as the IC50 in Table 10.

These data indicate that IC₅₀ values of RPPX at these receptors are approximately 290 to 649 times that of the IC₅₀ values for PPX. Further, these data suggest that the ratio of the IC₅₀ values for RPPX to PPX at the D₂ receptor are approximately 14 to 32 times larger than the ratios suggested by the literature, at least when the chiral purities were beyond the limits of detection (LOD 0.05%) (chiral purity greater than 99.95%). Similarly, the data suggest that the ratio of the IC₅₀ values for RPPX to PPX at the D₃ receptor are approximately 13 times larger than the ratios suggested by the literature, at least when the chiral purities were beyond the limits of detection (chiral purity greater than 99.95%). These data also suggest that if dopamine receptor affinity is the major contributing factor to limiting dose tolerance of the S(−) enantiomer, then pure preparations of the R(+) enantiomer should have a maximum tolerated dose (MID) and/or a no observable adverse effect level dose (NOAEL) of at least 290 times greater than the S(−) enantiomer's MTD and/or NOAEL. Thus, even a small contamination of the RPPX compositions of the present invention by the S(−) enantiomer, at levels as low as 0.5% or less, may effect the observed MID and NOEL.

Example 2

In vivo studies to determine the MTD and NOAEL in dogs for 100% pure IR preparations of the R(+) and S(−) enantiomers, and a mixture (R 99.5%/S 0.5%). The form of RPPX was RPPX dihydrochloride monohydrate.

The following in vivo study in beagle dogs was undertaken to test the hypothesis that the large observed difference in receptor binding affinities for the R(+) and S(−) enantiomers will translate to a large observed difference in the observed maximum tolerated dose (MTD) and/or no observable adverse effect level (NOAEL) of the two enantiomers. Dogs were administered IR preparations of each enantiomer prepared as a highly purified compound (100% pure preparations (within the limits of analytical detectability)), or a preparation of the R(+) enantiomer contaminated by 0.5% of the S(−) enantiomer.

Three groups of four non-naïve male beagle dogs were used in the study. Each group was administered various doses of either the R(+) or S(−) enantiomer prepared as a highly purified compound, or a preparation of the R(+) enantiomer contaminated by 0.5% of the S(−) enantiomer of 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole. Doses were administered orally by gavage and clinical observations were taken continuously following dosing: hourly for the first four hours, and then twice daily cage-side observations for the duration of the inter-dose or post-dose interval. Observations were made of clinical signs, mortality, injury and availability of food and water. Animals were fasted for 24 hr prior to dosing. Dogs in each group were exposed to only one drug, or the combination; each dose was administered only once, with a subsequent dose administered after a recovery period of 4 days. The data are summarized in Table 11.

A NOAEL was established at a dose level of 25 mg/kg for the R(+) enantiomer when administered to non-naïve dogs, while a dose level of 75 mg/kg may be considered an MTD in non-naïve dogs. For the S(−) enantiomer, a NOAEL of 0.00125 mg/kg and an MTD of 0.0075 mg/kg was found in non-naïve dogs. For the composition containing a mixture of the two enantiomers (99.5% RPPX and 0.5% PPX), the NOAEL was found to be 0.25 mg/kg, which corresponds to a dose of 00125 mg/kg of the S(−) enantiomer, while the MTD is 1.5 mg/kg, which corresponds to a dose of 0.0075 mg/kg of the S(−) enantiomer. These data indicate that the NOAEL for the R(+) enantiomer of 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole is approximately 20,000-fold greater than for the S(−) enantiomer in non-naïve dogs, while the MTD is about 10,000-fold greater.

TABLE 11 Clinical observations in male beagle dogs for administration of 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole compositions SUMMARY OF CLINICAL FINDINGS* Dose Amount (mg/kg) 7.5 25 75 0.0075 0.025 0.00125 1.5 5 0.25 R(+) R(+) R(+) S(−) S(−) S(−) mixture** mixture mixture (Day 1) (Day 4) (Day 8) (Day 1) (Day 4) Day 8) (Day 1) (Day 4) (Day 8) Behavior/Activity Activity decreased 0/4 0/4 2/4 3/4 4/4 0/4 4/4 4/4 0/4 Convulsions - clonic 0/4 0/4 1/4 0/4 0/4 0/4 0/4 0/4 0/4 Salivation 0/4 0/4 3/4 0/4 0/4 0/4 0/4 0/4 0/4 Tremors 0/4 0/4 4/4 1/4 3/4 0/4 1/4 2/4 0/4 Excretion Emesis 0/4 0/4 2/4 3/4 4/4 0/4 1/4 3/4 1/4 Feces hard 1/4 0/4 0/4 1/4 0/4 0/4 0/4 0/4 0/4 Feces mucoid 0/4 0/4 0/4 0/4 0/4 0/4 1/4 1/4 0/4 Feces soft 0/4 0/4 1/4 0/4 0/4 0/4 2/4 1/4 1/4 Feces watery 0/4 0/4 0/4 0/4 0/4 0/4 1/4 1/4 0/4 External Appearance Lacrimation 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 Eye/Ocular Pupils dilated 0/4 0/4 2/4 0/4 0/4 0/4 0/4 0/4 0/4 Pelage/Skin Skin warm to touch 1/4 0/4 1/4 0/4 0/4 0/4 0/4 0/4 0/4 *Number of animals affected/Total number of animals **Mixture of 99.5% RPPX and 0.5% PPX.

The data shown in Table 11 indicate that the receptor affinities identified (see Table 10) contribute in a straightforward fashion to the observed differences in the MTD and NOAEL doses for the R(+) and S(−) enantiomers of 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole. These data also indicate that the chiral purity for the R(+) enantiomer of pramipexole in embodiments of the compositions of the present invention (refer to Tables 5 and 6) may need to be in excess of 99.9%, depending on the final total dose, to avoid the adverse side effects of PPX.

Further, the data in Table 11 demonstrate that the NOAEL and MTD for the combination composition (99.5% RPPX and 0.5% PPX) may be determined directly by the dose of the S(−) enantiomer in the composition. Thus, a small (fractional percentage) contamination of a composition of RPPX by the PPX may reduce the MTD and NOEL of the composition. For example, in these experiments, the MTD of pramipexole was reduced from 75 mg/kg for the R(+) enantiomer to a total dose of 1.5 mg/kg of the mixed composition (a factor of 50), and the NOAEL was reduced from 25 mg/kg to 0.25 mg/kg, respectively (a factor of 100). Since the shift in MTD and NOAEL may be predicted by the dose of the S(−) enantiomer of 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole in the mixture, the shift for any unknown mixture may be calculated based on the percentage contamination of the RPPX by the PPX, relative to the MTD and NOAEL for PPX. This indicates that any contamination of an RPPX dosing solution with PPX will have a measurable effect on these indicators of dose tolerability.

Example 3.1 Toxicology Studies in Rats and Minipigs and Phase I Studies in Healthy Adult Volunteers.

Two-week and three-month toxicology studies of RPPX in rats and minipigs were completed. NOAEL dose levels of 150 mg/kg at two-weeks and 100 mg/kg at three-months for rats and 75 mg/kg at two-weeks and 50 mg/kg at three-months for minipigs were established. Phase I studies of healthy adult volunteers have demonstrated that RPPX in ascending single doses up to 300 mg and multiple doses up to 200 mg per day for 4½ days is safe and well-tolerated. The Mirapex® label specifies a starting dose of 0.125 mg and a maximum total daily dose of 4.5 mg. The Phase I data demonstrate, therefore, that RPPX may be safely administered (1) at starting doses that are at least 2400-fold higher than the Mirapex® starting dose and (2) at steady state doses that are at least 44-fold higher than the highest recommended dose of Mirapex®. The form of RPPX was RPPX dihydrochloride monohydrate.

The preliminary results of the clinical studies and the toxicology studies are discussed. Exposure at steady state in rats, minipigs, and humans is linear across all doses studied. After 3 months of dosing, the current No Observed Adverse Effect Level (NOAEL) in rats has been determined to be 100 mg/kg; and the current NOAEL in minipigs has been determined to be 50 mg/kg. The mean steady state AUC in rats at the NOAEL dose of 100 mg/kg was 61,299 and 61,484 h*ng/mL for males and females, respectively, and for minipigs at the NOAEL dose of 50 mg/kg was 91,812 and 131,731 h*ng/mL for males and females, respectively. The mean steady state AUC in humans at a dose of 100 mg Q12H (200 mg total daily dose) was 2,574 h*ng/mL. The drug has been safe, well-tolerated, and free of clinically significant adverse events in healthy adult subjects at single doses up to 300 mg and at multiple doses up to 100 mg Q12H, and the projected human exposure associated with a daily dose of 250 mg Q12H is expected to be greater than 13-fold lower than exposures seen at the NOAEL in male minipigs and approximately 9-fold lower than exposures seen at the NOAEL in male and female rats after 13 weeks of dosing.

3.2—Clinical Studies. RPPX has been studied at single daily IR doses of 50, 150 and 300 mg and twice daily IR doses of 50 and 100 mg for 4½ days in healthy adult volunteers. The drug has been safe and well-tolerated in both studies and there were no serious adverse events, discontinuations due to adverse events, or dose-related or clinically significant adverse events in either study. The most frequent adverse events have been dizziness and headache, all of which have been mild to moderate in severity and resolved without intervention.

3,2,1—Summary of (Blinded) Safety and Pharmacokinetic Results of RPPX (Ascending Single-Dose Study). Three sequential panels of 8 subjects each received single doses of RPPX (6 subjects) or placebo (2 subjects) at ascending JR dose levels of 50, 150, and 300 mg. Safety observations included vital signs, physical examination, clinical laboratory tests, ECGs, and adverse event reporting. Blood and urine samples were collected pre-dose and for 72 hours post-dose to assess the pharmacokinetics. All 24 subjects completed the study as planned. There were no serious adverse events; 46% of all subjects reported at least one non-serious adverse event (AE). Most AEs were mild; the most frequent AE was mild dizziness in 21% of subjects. There were no clinically significant safety observations at any dose level.

Pharmacokinetic data indicated that RPPX is rapidly absorbed with mean maximum concentrations of 125, 360, and 781 ng/mL reached at approximately 2 hours post-dose for the 50, 150, and 300 mg dose groups, respectively (see FIG. 1 and Table 12, below). Mean exposures (AUC_(0-∞)) were 1254, 3815, and 8623 h*ng/mL for the 50, 150, and 300 mg dose groups, respectively. Both C_(max) and AUC increased in proportion to dose across the dose levels tested. Urinary excretion of unchanged drug accounted for approximately 70% of drug elimination across dose levels. The mean T_(1/2) was 6-7 hours and was independent of dose. Comparison of the mean plasma concentrations (FIG. 2) and mean pharmacokinetic parameters (Table 12) after administration of a single 150 mg following a high fat/high calorie breakfast with those after administration of 150 mg under fasted conditions demonstrates essentially no effect of a meal on the absorption and elimination of RPPX.

Results of this study demonstrate that single JR oral doses of 50, 150, and 300 mg RPPX are safe and well-tolerated. The drug is orally bioavailable and the pharmacokinetics are linear. Absorption and elimination are not affected by a high fat/high calorie meal.

TABLE 12 Summary of pharmacokinetic parameters for RPPX after oral administration of single 50 mg, 150 mg, and 300 mg doses to healthy volunteers under fasted conditions and 150 mg under fed conditions. Fasted Fed Parameter¹ 50 mg 150 mg 300 mg 150 mg Cmax(ng/mL)  125 ± 22.0 (6)  360 ± 60.4 (6) 781 ± 158 (6) 315 ± 062 (6) Tmax(h) 2.04 (6) 2.04 (6) 1.96 (6) 2.58 (6) AUC(0−t) (h · ng/mL) 989 ± 295 (6) 3,387 ± 746 (6)   8,339 ± 3,202 (6) 3,099 ± 920 (6)   AUC(inf) (h · ng/mL) 1,254 ± 347 (6)   3,815 ± 972 (5)   8,623 ± 3,262 (6) 3,397 ± 944 (6)   λZ (h⁻¹) 0.1064 ± 0.0171 (6) 0.1001 ± 0.0087 (5) 0.1151 ± 0.0309 (6) 0.1152 ± 0.0256 (6) t½ (h) 6.65 ± 1.07 (6) 6.96 ± 0.56 (5) 6.40 ± 1.73 (6) 6.28 ± 1.48 (6) CL/F (mL/min) 706 ± 182 (6) 692 ± 183 (5) 659 ± 260 (6) 774 ± 165 (6) Vz/F (L)  395 ± 61.9 (6) 411 ± 081 (5)  346 ± 98.5 (6)  406 ± 62.8 (6) Ue (mg) 35.3 ± 5.19 (6) 60.5 ± 7.04 (6)  198 ± 28.0 (6) . ± . (0) Fe (% Dose) 70.7 ± 10.4 (6) 40.3 ± 4.69 (6) 65.8 ± 9.33 (6) . ± . (0) CLr (mL/min) 628 ± 149 (6)  310 ± 74.3 (6) 441 ± 159 (6) . ± . (0) ¹Mean ± standard deviation (N) except for Tmax for which the median (N) is reported.

3.2.2—Summary of (Blinded) Safety and Pharmacokinetic Results of RPPX (Ascending Multiple-Dose Study). This study is ongoing and has not yet been unblinded with respect to treatment assignments, and only clinical observations and pharmacokinetic data are available for the first 2 panels. To date, 2 sequential panels of 8 subjects each were enrolled to receive multiple doses of IR RPPX (6 subjects) or placebo (2 subjects). The first panel was administered a single IR dose of 50 mg, followed 48 hours later by 4½ days of multiple IR dosing (twice daily) at 50 mg Q12 hours. The second panel was administered a single IR dose of 100 mg, followed 48 hours later by 4½ days of multiple IR dosing (twice daily) at 100 mg Q12 hours. Safety observations included vital signs, physical examination, clinical laboratory tests, ECGs, and adverse event reporting. Blood samples were collected pre-dose on Day 1 and serially for 48 hours post-dose to assess the single-dose pharmacokinetics. Blood samples were collected pre-dose on Days 5, 6, and 7 to confirm steady-state was achieved, and serially through 72 hours post-dose on Day 7 to assess the steady-state pharmacokinetics of RPPX. Urine samples were collected for 12 hours after dosing on Day 7 to assess urinary excretion.

All 16 subjects enrolled to date have completed the study as planned. There were no deaths, reports of serious adverse events, or discontinuations because of adverse events during the study. Both dose levels were well tolerated. In cohort 1, all adverse events were mild in intensity, with the exception of moderate headaches reported by 2 subjects. In cohort 2, all adverse events were mild in intensity, with the exception of moderate “stiffness in back” and a moderate vasovagal response reported in 1 subject. An asymptomatic mild increase in heart rate upon standing (without change in blood pressure) was reported by the principal investigator for 1 of the 8 subjects dosed in cohort 1 (50 mg cohort) and for 2 of the 8 subjects dosed in cohort 2 (100 mg cohort). There were no clinically significant safety observations at any dose level.

Pharmacokinetic data are shown in Table 13 and FIG. 3. C_(max) and AUC₍₀₋₁₂₎ increased 37% and 40%, respectively from Day 1 to Day 7 for subjects receiving 50 mg Q12H, with essentially no change in T_(max). Mean exposure AUC₍₀₋₁₂₎ at Day 7 was 1449 h*ng/mL for the 50 mg Q12H dose group. C_(max) and AUC₍₀₋₁₂₎ increased 24% and 38%, respectively from Day 1 to Day 7 for subjects receiving 100 mg Q12H, with essentially no change in T_(max). Mean exposure AUC₍₀₋₁₂₎ at Day 7 was 2465 h*ng/mL for the 100 mg Q12H dose group. Results of this study demonstrate that multiple oral doses of 50 and 100 mg RPPX administered twice daily are safe and well-tolerated. The drug is orally bioavailable and the pharmacokinetics are linear at steady state, with no significant accumulation.

TABLE 13 Summary of pharmacokinetic parameters for RPPX during oral administration of 50 mg and 100 mg IR doses on Day 1, Q12H on Days 3 through 6, and a single dose on Day 7 to healthy volunteers under fasted conditions. Dose Parameter¹ 50 mg 100 mg Day 1 Cmax (ng/mL)  139 ± 15.3 (6)  248 ± 30.4 (6) Tmax (h) 1.83 (6) 1.92 (6) AUC(0−12) (h · ng/mL) 1,035 ± 121 (6)   1,776 ± 260 (6)   AUC(0−t) (h · ng/mL) 1,463 ± 280 (6)   2,545 ± 497 (6)   AUC(inf) (h · ng/mL) 1,502 ± 280 (6)   2,574 ± 505 (6)   λz (h⁻¹) 0.1132 ± 0.0230 (6) 0.1073 ± 0.0161 (6) t½ (h) 6.34 ± 1.31 (6) 6.57 ± 0.88 (6) CL/F (mL/min) 571 ± 107 (6) 665 ± 107 (6) Vz/F (L)  306 ± 45.8 (6)  373 ± 51.0 (6) Day 7 Cmax (ng/mL)  191 ± 20.9 (6) 306 ± 055 (6) Tmax (h) 1.75 (6) 2.00 (6) AUC(0-12) (h · ng/mL) 1,449 ± 221 (6)   2,465 ± 299 (6)   λz (h⁻¹) 0.1025 ± 0.0186 (6) 0.0894 ± 0.0117 (6) t½ (h) 6.96 ± 1.30 (6) 7.88 ± 1.19 (6) CL/F (mL/min)  585 ± 81.6 (6)  684 ± 76.1 (6) Vz/F (L)  346 ± 30.1 (6)  466 ± 82.2 (6) Ue (mg) . ± . (0) . ± . (0) Fe (% Dose) . ± . (0) . ± . (0) CLr (mL/min) . ± . (0) . ± . (0) ¹Mean ± standard deviation (N) except for Tmax for which the median (N) is reported.

3.3—Toxicology Studies. In a 2-week repeat-dose toxicology studies in rats, animals received 50, 150, and 500 mg/kg IR doses of RPPX for 14 days. RPPX caused mortality at the high dose of 500 mg/kg and statistically significant changes in body weight gain and food consumption for both sexes were observed in the animals surviving to terminal sacrifice. No target organ toxicity by histopathology examination was identified at any dose. The NOAEL for this 2-week study in rats was determined to be 150 mg/kg. Following this study, 3- and 6-month repeat dose toxicology studies were completed at doses of 30, 100, and 300 mg/kg. The results of the 3-month study contain some target organ toxicity by histopathology examination at the highest dose (300 mg/kg) with no test article related deaths and no significant clinical observations outside of several incidences of convulsions in high dose rats lasting approximately 2 minutes. The animals' health did not appear to be otherwise adversely affected by these convulsions. Test article-related microscopic changes were observed in the liver (minimal grade cholestasis correlating with increased total bilirubin), ileal small intestine (minimal grade mineralization), and thymus (minimal grade lymphoid depletion correlating with lower group thymus weights compared to controls). The NOAEL for the 3-month study in rats is considered to be 100 mg/kg. Systemic exposure (AUC_(0-last)) at week 13 at the NOAEL dose of 100 mg/kg was 61,299 h*ng/mL in males and 61,484 h*ng/mL in females. The in-life phase of the 6-month toxicology study in rats was recently completed and histopathologic examinations are pending. There were no mortalities at any dose level between the 13-week and 26-week sacrifices.

In a 2-week repeat-dose toxicology study in minipigs, animals received 7.5, 25 and 75 mg/kg IR doses of RPPX for 14 days. No target organ toxicity by histopathology examination was identified at any dose. Clinical observations included salivation, decreased activity, emesis and inappetance, with higher incidences of emesis in females than males, and mostly in the 75 mg/kg group. The incidence of emesis at 75 mg/kg (at least one episode in 5 of 8 animals dosed at 75 mg/kg) suggested this dose is close to the limit of tolerability for RPPX for chronic dosing in minipigs. Since no test article related toxicological changes were observed at the high dose, the NOAEL for the 2-week study was considered to be greater than or equal to 75 mg/kg. Based on this study, 3-, and 6-, and 9-month repeat dose studies of RPPX in minipigs were initiated at dose levels of 7.5, 25 and 75 mg/kg. At month 2, dose levels were reduced to 7.5, 25 and 50 mg/kg due to mortalities at the 75 mg/kg level. The 3- and 6-month repeat dose studies have now been completed at the 7.5, 25 and 50 mg/kg dose levels and the 9-month repeat dose study is ongoing. No target organ toxicity by histopathology examination was identified at any dose level following animal sacrifice after 3 months of exposure. The NOAEL for the 3-month study in minipigs is considered to be 50 mg/kg. Systemic exposure (AUC0-24) at week 13 at the NOAEL dose of 50 mg/kg/day was 91,812 h*ng/mL in males and 131,731 h*ng/mL in females. The in-life phase of the 6-month toxicology study in minipigs was recently completed and histopathologic examinations are pending. There were no mortalities attributed to test article or significant clinical observations at any dose level between the 13-week and 26-week sacrifices. The ongoing 9-month toxicology studies in minipigs have now passed month 7 and no deaths attributed to test article or significant clinical observations have occurred at any dose level.

3.4. Human Dosages. The development of RPPX as a treatment of ALS is based on a maximally tolerated dose strategy, derived either from tolerability or safety data from studies in humans or from the results of animal toxicology studies. To date there have been no dose-limiting tolerability observations in humans. Therefore, in order to progress dosing in humans, it is necessary to closely examine the exposure at which toxicity has been observed in rats and minipigs. Pharmacokinetic data obtained to date suggest that pharmacokinetics in humans will continue to be dose-proportional at higher doses, and that the accumulation factor will be constant. Safety and toxicokinetic results from the 3 month toxicology studies in rats and minipigs show no adverse effects of chronic dosing up to 100 mg/kg in rats and 50 mg/kg in minipigs. Analysis of safety margins in RPPX exposure between the NOAEL for minipigs and the projections of human exposure, therefore, support progression of total daily doses up to at least 500 mg in humans. The projected steady-state exposure of RPPX at a total daily dose of 500 mg administered as 250 mg Q12H is approximately 7,000 h*ng/mL, which is greater than 13-fold lower than exposures seen at the NOAEL in male minipigs and approximately 9-fold lower than exposures seen at the NOAEL in male and female rats after 13 weeks of dosing.

FIGS. 4 and 5 are plots of exposure vs. dose for rats and minipigs, respectively, compared with humans. Each graph displays the relationship between exposure as expressed by AUC (h*ng/mL) and dose as expressed by body surface area (mg/m2) at every dose level administered to each species in both the 2-week and 13-week assessments. Individual data points with error bars are the mean±SD. The dashed horizontal line at the bottom of both charts illustrates the extrapolated steady state AUC (7,000 h*ng/mL) in humans at 250 mg Q12H. Table 17A and Table 17B are an integrated summary of all human pharmacokinetic estimates obtained in the two Phase I studies.

TABLE 17A Summary of the human pharmacokinetic estimates obtained in the two Phase I studies with healthy volunteers Dose Dosing Cmax Tmax AUC(0-t) AUC (inf) AUC(0−12) Study (mg) Regimen Food (ng/mL) (h) (h*ng/mL) (h*ng/mL) (h*ng/mL) CL001 50 SD Fasted 125 ± 22.0 2.04 (6)   989 ± 295 1,245 ± 347 — (6) (6) (6) 150 SD Fasted 360 ± 60.4 2.04 (6) 3,387 ± 746 3,815 ± 972 — (6) (6) (5) 300 SD Fasted 781 ± 158  1.96 (6)   8,339 ± 3,202   8,623 ± 3,262 — (6) (6) (6) 150 SD Fasted 315 ± 062  2.58 (6) 3,099 ± 920 3,397 ± 944 — (6) (6) (6) CL002 50 Q12H Fasted 139 ± 15.3 1.83 (6) 1,463 ± 280 1,502 ± 280 1,035 ± 121 (Day 1) (6) (6) (6) (6) (Day 7) Fasted 191 ± 20.9 1.75 (6) — — 1,449 ± 221 (6) (6) 100 Q12H Fasted 248 ± 30.4 1.92 (6) 2,545 ± 497 2,574 ± 505 1,776 ± 260 (Day 1) (6) (6) (6) (6) (Day 7) Fasted 306 ± 055  2.00 (6) — — 2,465 ± 299 (6) (6) 250 Q12H Fasted — — — — — (Day 1) (Day 7) Fasted — — — — — Mean ± standard deviation (N) except for T_(max) for which the median (N) is reported. SD = single dose

TABLE 17B Summary of the human pharmacokinetic estimates obtained in the two Phase I studies with healthy volunteers (continued) Dose Dosing t½ CL/F Vz/F Ue CLx Study (mg) Regimen Food (h) (mL/h) (L) (mg) (% Dose) (mL/min) CL001 50 SD Fasted 6.65 ± 1.07 706 ± 182 395 ± 61.9 35.3 ± 5.19   70.7 = 10.4 628 = 149 (6) (6) (6) (6) (6) (6) 150 SD Fasted 6.96 ± 0.56 692 ± 183 411 ± 081  60.5 ± 7.04   40.3 = 4.69  310 = 74.3 (5) (5) (5) (6) (6) (6) 300 SD Fasted 6.40 ± 1.73 659 ± 260 346 ± 98.5 198 ± 28.0   65.8 = 9.33 441 = 159 (6) (6) (6) (6) (6) (6) 150 SD Fasted 6.28 ± 1.48 774 ± 165 406 ± 62.8 . ± (0) . ± (0)   . ± (0) (6) (6) (6) CL002 50 Q12H Fasted 6.34 ± 1.31 571 ± 107 306 ± 45.8 — — — (Day 1) (6) (6) (6) (Day 7) Fasted 6.96 ± 1.30  585 ± 81.6 346 ± 30.1 . ± (0) . ± (0)   . ± (0) (6) (6) (6) 100 Q12H Fasted 6.57 ± 0.88 665 ± 107 373 ± 51.0 — — — (Day 1) (6) (6) (6) (Day 7) Fasted 7.88 ± 1.19  684 ± 76.1 466 ± 82.2 . ± (0) . ± (0)   . ± (0) (6) (6) (6) 250 Q12H Fasted — — — (Day 1) (Day 7) Fasted — — — Mean ± standard deviation (N) except for T_(max) for which the median (N) is reported. SD = single dose

Exposure at steady state in rats, minipigs, and humans is linear across all doses studied. After 3 months of dosing, the NOAEL in rats has been determined to be 100 mg/kg; and the NOAEL in minipigs has been determined to be 50 mg/kg. The mean AUC in rats at the NOAEL was 61,299 and 61,484 h*ng/ml, for males and females, respectively, and for minipigs was 91,812 and 131,731 h*ng/mL for males and females, respectively. The mean AUC in humans at steady state at a dose of 100 mg Q12H (200 mg total daily dose) was 2,574 h*ng/mL.

Example 4 Preparation of Capsules with RPPX

RPPX dihydrochloride monohydrate is filled in hard gelatin capsules with no excipients. The capsules used for the drug product are #00 blue opaque gelatin capsules from Hawkins Chemical Group. Dose strengths of 50 and 500 mg are produced. Matching placebo capsules are filled with microcrystalline cellulose. Capsules are prepared by weighing individual empty capsules and recording the weight (W_(e)). Specified amount of active drug substance are individually weighed and hand-filled into a capsule bottom using a Torpac® filling funnel. A purity adjustment factor of 1.0638 is used to adjust for the water weight (monohydrate) in the salt form, i.e., a 50 mg dose should have a target fill of 50×1.0638=53.16 mg. Capsule tops are joined with the filled capsule bottom. The filled capsules are then weighed, and the weight is recorded (W_(f)). The calculated weight of the drug substance in the capsule (W_(f)−W_(e)) is recorded. If this calculated weight is within +/−5% of the nominal weight, then the capsule is cleaned, polished, and placed into and appropriately labeled container. If the calculated weight is outside of the specified range, the capsule is discarded. The free-base weight per capsule (free-base weight per mg of capsule contents multiplied by fill weight) is 90% to 100% of the calculated label claim. Total impurities are ≦2%. The appearance is a blue capsule containing white to off-white powder.

Example 4B Preparation of Tablets with RPPX

Capsules with 125 mg dose strength are prepared with the composition shown in Table 17. Capsules are generally prepared under conditions of 60 to 74° F. and a relative humidity of 30 to 60%. Microcrystalline cellulose, mannitol, crospovidone, magnesium stearate, and RPPX (milled) are weighed out in the amounts shown in the column “Quantity/batch” in Table 14. The microcrystalline cellulose, mannitol, crospovidone, and RPPX are then hand screened through a #20 mesh stainless steel screen and transferred to a Maxiblend V-blender with a 4 quart shell. The materials are then mixed using the Maxiblend V-blender for 10 minutes. The magnesium stearate is then screened using a 30 mesh stainless steel hand screen and transferred to the blender. The powders are then mixed for five minutes. The final blend is then emptied into a labeled, double PE-lined drum and the gross, tare, and net weights are recorded.

Tablets are prepared using a Minipress II B with 5 stations of ⅜″ round, standard, concave tooling and gravity feed frame. The final blend is placed in the hopper and the tablet press set up is run according to the specifications in Table 15.

TABLE 14 Tablet and Batch Compositions Quantity/ Quantity/ Ingredient Percent unit (mg) batch (g) RPPX (milled) 40.00 125.00 400.000 Microcrystalline cellulose (Avicel 35.25 110.16 352.512 PH102) (Diluent) Mannitol (Pearlitol SD100) (Diluent) 20.00 62.50 200.000 Crospovidone (Polyplasdone XL) 4.00 12.50 40.000 (Disintegrant) Magnesium stearate (vegetable source, 0.75 2.34 7.488 grade 905-G) (Lubricant) Total 100.00 312.50 1000.000

TABLE 15 Tablet Press Settings Parameter Target (range) Average tablet weight 3.125 g (3.031 g to 3.219 g) (+/−3%) (10 tablets) Target weight 312.5 mg (296.9 mg to 328.1 mg) (+/−5%) (individual tablet) Target hardness 12 Kp (6 Kp to 18 Kp) Press speed 20 rpm (10 to 30 rpm)

Example 5 Preparation of RPPX P-TSA Salt: Condition A:

All reagents were purchased from CNH technologies, Fisher, Aldrich, G.J. Chemicals, Puritan, TCI and Spectrum and were used as provided. Proton nuclear magnetic resonance spectra were obtained on a Bruker AC 300 spectrometer at 300 MHz. HPLC analysis for chiral purity was performed on a Chiralpak® IA column (5 μM, 250×4.6 mm) at 30° C. using a mobile phase of heptane/ethanol/diethylamine (80:20:2 v/v/v). HPLC analysis for chemical purity was performed on a Sunfire® column (3.5 μM, 150×4.6 mm) at 30° C. using two mobile phases: A—0.5% TFA in water; and B—0.5% TFA in methanol. A gradient of 5% B to 80% B was used to separate the diamine and pramipexole peaks. A detection wavelength of 265 nm was used for both HPLC analyses.

Each of the processes detailed in examples 5-14 may also be scaled for industrial manufacturing processes, as shown in examples 15-17. Certain examples have been detailed at both the laboratory scale and the industrial manufacturing scale to demonstrate that the chemical and chiral yields are independent of the scale of the synthesis.

A 2.0 liter, three-necked flask was equipped with an overhead stirrer, a temperature probe, a heating mantle, a claisen joint, a reflux condenser, and a 500 ml addition funnel. The flask was charged with 45 grams of R(+)-2,6 diamino-4,5,6,7-tetrahydro-benzothiazole, followed by 750 ml of n-propanol. Under continuous stirring, the mixture was heated to a temperature of 95° C. over 15 minutes generating a clear solution. The addition funnel was charged with a solution of 74 grams propyl tosylate and 60 ml diisopropylethyleamine in 250 ml n-propanol. This solution was added dropwise to the 2.0 liter flask with continuous stirring over a period of 4 hours. The reaction was continued with stirring for an additional 8 hours at 95° C., after which the solution was brought to room temperature, and stirring was continued for an additional 4 hours.

The precipitated material was collected by filtration and washed three times using 100 ml reagent grade alcohol each time. The alcohol washed precipitated cake was then washed with 100 ml heptane and dried under high vacuum for 2 hours. The final weight of the dried product was 53.2 grams, representing a 52.2% yield. HPLC was used to determine the chemical purity of the R(+)-2,6-diamino-4,5,6,7-tetrahydro-benzothiazole (RPPX) as 98.2% and the chiral purity as greater than 99.5%. ¹H NMR and ¹³C NMR were used to confirm the structure.

Example 6 Preparation of Racemic Pramipexole P-TSA Salt: Condition A

A 250 ml, three necked flask was equipped with a magnetic stirrer, a temperature probe, a heating mantle, a claisen joint, a reflux condenser, and a 100 ml addition funnel. The flask was charged with 5 grams of racemic 2,6 diamino-4,5,6,7-tetrahydro-benzothiazole, followed by 80 ml of n-propanol. Under continuous stirring, the mixture was heated to a temperature of 95° C. over 15 minutes generating a clear solution. The addition funnel was charged with a solution of 10.12 grams propyl tosylate and 9.2 ml diisopropylethyleamine in 28 ml n-propanol. This solution was added dropwise to the 250 ml flask with continuous stirring over a period of 2 hours. The reaction was continued with stirring for an additional 6 hours at 95° C., after which the solution was brought to room temperature, and stirring was continued for an additional 6 hours.

The precipitated material was collected by filtration and washed two times using 25 ml reagent grade alcohol each time. The alcohol washed precipitated cake was then washed with 25 ml heptane and dried under high vacuum for 1 hours. The final weight of the dried product was 5.12 grams, representing a 45% yield. HPLC was used to determine the chemical purity of the racemic 2,6-diamino-4,5,6,7-tetrahydro-benzothiazole (racemic pramipexole) as 97.12%, and the chiral purity showed a 1:1 mixture of the R(+) and PPX. ¹H NMR was used to confirm the structure.

Example 7 Preparation of RPPX P-TSA Salt: Condition C

A 12 L, three necked flask was equipped with an overhead stirrer, a temperature probe, a heating mantle, a claisen joint, a condenser, and a 500 ml addition funnel. The flask was charged with 250 grams of R(+)-2,6 diamino-4,5,6,7-tetrahydro-benzothiazole (R(+) diamine), followed by 2 L of dimethyl formamide (DMF). Under continuous stirring, the mixture was heated to a temperature of 65° C. The addition funnel was charged with a solution of 386.6 grams propyl tosylate (1.25 molar equivalents) and 322 ml diisopropylethyleamine (1.25 molar equivalents) in 500 ml DMF. This solution was added to the 12 L flask dropwise over a period of 2.0 hours. The reaction was monitored by analysis on HPLC.

The reaction was continued at 65° C. for an additional 5 hours, after which the solution was gradually cooled to room temperature and stirred overnight. The solution was diluted with 2 L MTBE and stirred for an additional 0.5 hours. The precipitated material was collected by filtration and washed with 500 ml MTBE, followed by 3 washes of 500 ml each reagent alcohol. The washed precipitated cake was dried under high vacuum.

The final weight of the dried product was 317.6 grams, representing a 56% yield. HPLC was used to determine the chemical purity of the R(+)-2,6-diamino-4,5,6,7-tetrahydro-benzothiazole (PPX) as 98.4% and the chiral purity as greater than 99.8%. ¹H NMR and ¹³C NMR was used to confirm the structure. ¹H NMR (300 MHz, DMSO-d6) δ 8.5 (br.s, 2H), 7.5 (d, 2H), 71.2 (d, 1H), 6.8 (s, 2H), 3.4 (m, 1H), 2.95 (m, 3H), 2.6 (m, 2H, merged with DMSO peak), 2.3 (s, 3H), 2.15 (m, 1H), 1.8 (m, 1H), 1.55 (m, 2H), 0.9 (t, 3H); ¹³C NMR (300 MHz, DMSO-d6) δ 167.0, 145.5, 144.6, 138.4, 128.6, 125.8, 110.7, 53.9, 46.5, 25.8, 25.6, 24.5, 21.2, 19.6, 11.3.

Example 8 Conversion of RPPX P-TSA Salt to RPPX Dihydrochloride

RPPX p-TSA salt (50 grams; 0.13 mol) was taken into 150 ml absolute ethanol and cooled to between 0 and 5° C. with continuous stirring. Concentrated HCl (33 ml) was slowly added to the reaction while maintaining the temperature at between 0 and 5° C., and the mixture was stirred for an additional 15 minutes. MTBE (200 ml) was added to the mixture, and stirring was continued for an additional 1.5 hours at temperature. The reaction mixture was then filtered, washed twice with an MTBE/ethanol solution (2:1, 2×50 ml wash volumes), and dried under vacuum at 30° C. overnight. The final product was 34 grams of RPPX dihydrochloride, indicative a of 92% yield, and a 97.3% chemical purity as determined by HPLC.

Example 9 Conversion of RPPX P-TSA Salt to RPPX Dihydrochloride

RPPX p-TSA salt (10 grams; 0.026 mol) was dissolved in 200 ml IPAC and cooled to 15° C. with continuous stirring. HCl gas was bubbled into the slurry for 1 hour. The mixture was then filtered, washed with IPAC, and dried overnight under vacuum at room temperature. The final product was 6.8 grams of RPPX dihydrochloride, indicative a of 92% yield, and a 97% chemical purity as determined by HPLC.

Example 10 Conversion of RPPX P-TSA Salt to RPPX Free Base

RPPX p-TSA salt (25 grams; 0.065 mol) was dissolved in 200 ml DCM and mixed into a slurry. 10 ml of water was added and the mixture was basified with 12 ml of 6N NaOH to a pH of 11-12. The two phases were split, and the aqueous was extracted with 200 ml of DCM. The combined organic phases were dried over MgSO₄, filtered over Celite® and concentrated. The residue was dissolved in 100 ml MTBE and slurried for several hours. The solids were then filtered, washed with MTBE and dried under vacuum at 35° C. The final product was 9.1 grams of RPPX dihydrochloride, indicative a of 66% yield, and a 98% chemical purity as determined by HPLC.

Example 11 Conversion of RPPX P-TSA Salt to RPPX Free Base

Freebase formation was performed on a 200 gram scale. A 5 L, three necked, round-bottomed flask, equipped with an over head stirrer, thermometer, and addition funnel was charged with 200 g (0.522 mol) of RPPX p-TSA salt and 1 L of water. The mixture was stirred and cooled to 10° C. The slurry was basified to a pH of about 11-12 by the slow addition of 200 ml of 6 N NaOH over period of 15 min. The reaction mixture was diluted with 500 ml of brine (sodium chloride dissolved in water) and extracted with 3×1 L of dichloromethane. The combined organic phases were washed with 1.0 L of brine, dried over MgSO₄, filtered and concentrated to dryness. The residue was triturated with 1 L of 1:1 IPAC:Heptane, the resulting slurry was stirred for 1 hour, filtered and the filter cake was washed with 2×250 ml of 1:1 mixture of IPAC:Heptane, The filter cake was collected and dried at 40° C. under high vacuum for 24 hours to give 94.1 grams RPPX (85.5%) as a white solid. The chemical purity was 100% AUC as tested by HPLC, and the chiral purity was 100% AUC as tested by HPLC. ¹H NMR and ¹³C NMR was used to confirm the structure: ¹H NMR (300 MHz, DMSO-δ6) δ 6.6 (s, 2H), 2.8 (m, 2H), 2.5 (m, 2H, merged with DMSO peak), 2.2 (m, 1H), 1.9 (m, 1H), 1.5-1.3 (m, 4H), 0.85 (t, 3H); ¹³C NMR (300 MHz, DMSO-d6) δ 166.2, 144.8, 113.6, 54.2, 49.1, 30.0, 29.6, 25.2, 23.5, 12.3.

Example 12 Conversion of RPPX Free Base to RPPX Dihydrochloride

The freebase of RPPX (4.8 grams; 0.022 mol) was dissolved in 200 ml of IPAC and cooled to 15° C. HCl gas was bubbled into the slurry for 1 hour. The mixture was then filtered, washed with IPAC and dried under vacuum at room temperature overnight. The final product was 6.4 grams of RPPX dihydrochloride, indicative a of 100% yield, and a 97% chemical purity as determined by HPLC.

Example 13 Conversion of RPPX Free Base to RPPX Dihydrochloride

The freebase of RPPX (50 grams; 0.13 mol) was dissolved in 500 ml of IPAC. Under continuous stirring, the mixture was slowly charged with 78 ml of concentrated HCl at a temperature of 25° C. The mixture was stirred overnight at ambient conditions (˜25° C.), filtered and dried under vacuum at 40° C. The final product was 68 grams of RPPX dihydrochloride, indicative a of 95% yield.

Example 14 Optical Purification of RPPX Using Achiral Acid Addition

Pramipexole enantioenriched for the R(+) enantiomer (300 mg) was dissolved in 10 ml of the chosen solvent at 75° C. (see examples in Table 8; ethanol or acetonitrile). Complete dissolution was observed in all samples. Acid addition was made at 1.05 molar equivalents for the p-TSA (solvent is ethanol; 2.97 ml of 0.5 M acid) and MSA (solvent is acetonitrile; 1.49 ml of 1.0 M acid), and 2.05 molar equivalents for the fumaric (solvent is acetonitrile; 5.84 ml of 0.5 M acid) and phosphoric (solvent is acetonitrile; 2.90 ml of 1.0 M acid). The reaction mixtures were cooled to room temperature at a rate of 25° C./hour and stirred at room temperature for an additional 19 hours. The solids obtained by this trituration step were isolated by filtration and dried under high vacuum at room temperature. These products were analyzed by HPLC, ¹H NMR, thermal gravimetric analysis, differential scanning calorimetry, X-ray powder diffraction (XPRD), Fourier transform infrared spectroscopy and moister-sorption analysis. The XPRD patterns showed that the p-TSA, MSA and fumarate salt forms of the RPPX were crystalline, while the phosphate salt form of the RPPX was amorphous.

Example 15 Industrial Scale Resolution of Racemic Diamine

A 72 L, unjacketed reactor was charged with racemic 2,6 diamino-4,5,6,7-tetrahydro-benzothiazole (rac-diamine) (4.5 kg; 26.6 mol) and 58.5 L water, and heated as a suspension to a temperature of about 60° C. to 65° C. Resolution of the enantiomers was achieved by addition of one equivalent of (D)-(−)-Tartaric acid (3991 grams; 26.6 mol) in 4.5 L of water, after which the resulting solution was heated to a temperature of about 70° C. to 75° C. and maintained at this temperature for about 1 hour. The mixture was allowed to cool to a temperature of about 20° C. to 25° C. and stirred for an additional 15 hours, after which the mixture was filtered and the solids were washed 3× with water (6.3 L each wash).

The wet solids, which contain the R(+) enantiomer of the diamine, were charged to the reactor followed by 54 L of water, and the mixture was heated to a temperature of about 70° C. to 75° C. for 2 hours. The mixture was allowed to cool to a temperature of about 20° C. to 25° C. and stirred for 17 hours. The mixture was then filtered and the solids were washed 2× with water (4.5 L each wash). The wet solids were transferred to a jacketed reactor and the reactor was charged with 8.1 L of water. The mixture was cooled to a temperature of about 0° C. to 5° C. and cautiously charged with concentrated 1.625 L of HCl, followed by 1.155 L of 50% NaOH to achieve a pH of about 9-10. During the addition the temperature was maintained at about 0° C. to 506, and stirred for an additional hour at temperature. The resulting mixture was then filtered and the solids were washed 2× with cold (0° C. to 5° C.) water (1.125 L each wash). The solids were transferred to a jacketed reactor and were reslurried once more with 4.5 L of water at 0° C. to 5° C. The solids were filtered and dried under warn air (40° C. to 45° C.) to give 1940 grams of the product (R(+) diamine) as a white solid, with an 86% yield for the R(+) enantiomer.

The mother liquors of the initial resolution step, which contain the S(−) enantiomer of the diamine, were concentrated to afford diamine with a 95.5% yield for the S(−) enantiomer.

TABLE 16 Experiments for industrial scale resolution of the R(+) enantiomer of diamine Yield (%) Input of R(+) Chemical Purity Chiral Purity (grams) enantiomer (AUC % by HPLC) (AUC % by HPLC) 1000 76 >99 98.3 4500 86 >99 98.5 4100 54 >99 98.5

Example 16 Industrial Scale Preparation of Propyl Tosylate

A 100 L glass, jacketed reactor was charged with 1-propanol (2.098 kg; 34.9 mol), triethylamine (4.585 kg; 45.3 mol; 1.3 equivalents) and DCM (20.1 L). The mixture was cooled to a temperature of about 5° C. to 15° C. and cautiously charged with a solution of p-toluenesulfonyl chloride (6 kg; 31.47 mol; 0.9 equivalents) in DCM (10.5 L) over 30 minutes. Once the addition was complete, the mixture was warmed to a temperature of about 18° C. to 22° C. and stirred for 12 hours. The reaction mixture was assayed by ¹H NMR (in CDCl₃) and deemed complete. HCl (6 N; 2.98 L) was cautiously charged while maintaining the temperature below 25° C. The aqueous phase was removed, and the organic phase was washed 2× with water (21 L each wash), dried with MgSO₄, and filtered over Celite®. The filtered solids were then washed with DCM (4 L) and concentrated to a residue. The residue was dissolved in heptane and concentrated again to afford a final propyl tosylate product (6.385 kg, 95% yield).

The present invention provides evidence that the dopamine receptor affinity of RPPX is actually much lower than previously appreciated. In a study using beagle dogs presented herein, it has been shown that the functional separation between the PPX and RPPX enantiomers (10.000-20,000 fold) is much greater than previously expected. These data also show that contamination of the composition of pure RPPX with small, known amounts of PPX results in a predictable shift in the MTD of the composition. These data demonstrate that RPPX can be dosed at levels that can more fully and unexpectedly exploit the lower-potency neuroprotective potential of the compound without the theoretical MTD limitation previously assumed, and without the need for dose titration. The application presents methods for using pure compositions of RPPX in acute and chronic neurodegenerative disorders previously inaccessible to this drug and immediately at full-strength without dose-titration and at higher theoretical MTDs. Additionally, the data showing that a pure composition of RPPX can be mixed with a known amount of PPX to produce dopamine receptor agonist effects determined solely by the contribution of the (S)-enantiomer allows for the use of compositions comprising the mixture of known amounts of pure (R)- and (S)-enantiomers for use in neurodegenerative disorders amenable to both dopamine receptor agonist treatment and neuroprotection, such as PD.

Example 17 Industrial Scale Preparation of RPPX P-TSA Salt: Condition C

A 72 liter unjacketed reactor was charged with 1.84 kg (10.87 mol) of R(+)-2,6 diamino-4,5,6,7-tetrahydro-benzothiazole (R(+) diamine), followed by 14.7 L of dimethyl formamide (DMF). Under continuous stirring, the mixture was heated to a temperature of between 65° C. and 68° C. A solution of 2926 grams propyl tosylate and 1761 grams diisopropylethyleamine in 3.455 L DMF was added slowly over a period of 2 hours. The reaction was continued at 67° C. for an additional 4 hours, after which the solution was gradually cooled to room temperature (18° C. to 22° C.) and stirred for an additional 15 hours. The solution was diluted with 14.72 L of MTBE over a time period of 30 minutes, and stirred for an additional 1 hour. The precipitated material was collected by filtration and washed with 7.32 L, MTBE, followed by 3 washes of 3.68 L each of ethanol, and a wash with 9.2 L heptane. The washed precipitated cake was dried under high vacuum at 30° C. to 35° C. The final weight of the dried product was 2090 grams, representing a 50% yield.

Example 18

The following provides an exemplary simulation of the plasma concentration-time profiles of KNS-760704 that would be obtained at steady-state after administration according to various regimens based on a compartmental model that was fit to the mean data from Study KNS-760704-CL001, in which single 50 mg and 150 mg doses were administered to healthy volunteers, wherein the target was a trough concentration of 1 μM (211 ng/mL) with once-daily dosing of 300 mg RPPX as the salt (equivalent to 224 mg base) in a matrix-type formulation with substantially first-order type of release.

The predicted steady-state plasma concentration-time profile obtained after systematic variation of the absorption rate is shown in FIG. 6. This profile, which has highest Cmin that could be obtained with a 300 mg dose, 185 ng/mL (0.9 μM), was obtained with an apparent first-order absorption rate constant of 0.02 h⁻¹, assumed to be “equal” to the in vitro release rate. Slower input rates resulted in a decrease in plasma concentrations due to the reduced amount of drug that could be input over the 24-hour period. FIG. 7 contains the corresponding release profile; assuming a Level A in vitro/in vivo correlation, this would also represent the in vitro dissolution profile for the formulation, and the percent released at the times normally used in dissolution testing are shown in Table 17 below.

TABLE 17 Predicted in vivo/in vitro release profile for a once-daily controlled-release KNS-760704 formulation. Time (h) Percent Released 1 2 2 4 3 6 4 8 8 15 12 20 18 30 24 40

The modeling predicts a Cmin of 185 ng/mL and a Cmax of 219 ng/mL, slightly under and over the 211 ng/mL (1 μM) target trough and provides a relatively flat curve. This modeling provides just one example of a particular extended release formulation. Other formulations, as described herein, can be formulated to provide/maintain other targeted Cmin and Cmax measurements. For example, a formulation comprising 400 mg that may result in a Cmin of about 211 ng/mL (1 μM) and a Cmax of about 320 ng/mL, well below the observed Cmax of about 479 ng/mL after administration of the immediate release 150 mg formulation every 12 hours in KNS-760704-CL002.

Although the present invention has been described in considerable detail with reference to certain preferred embodiments thereof, other versions are possible. Therefore the spirit and scope of the appended claims should not be limited to the description and the preferred versions contained within this specification. 

What is claimed is:
 1. A method of treating a chronic neurodegenerative disease selected from the group consisting of: Parkinson's disease and amyotrophic lateral sclerosis, in a patient in need thereof comprising administering a single unit dose of about 600 milligrams to about 1,000 milligrams of (6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine in a modified release pharmaceutical composition wherein the modified release composition releases the (6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine over a 24 hour period.
 2. The method of claim 1, wherein said patient is a naïve patient.
 3. The method of claim 1, wherein said chronic neurodegenerative disease is amyotrophic lateral sclerosis.
 4. The method of claim 1, wherein the chiral purity for the (6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine is 99.9% or greater.
 5. The method of claim 1, wherein the chiral purity for the (6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine is 99.95% or greater.
 6. The method of claim 1, wherein the chiral purity for the (6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine is 99.99% or greater.
 7. A method of treating a neurodegenerative disease selected from the group consisting of: Parkinson's disease and amyotrophic lateral sclerosis, in a patient in need thereof comprising administering to said patient a starting dose of about 600 milligrams to about 1,000 milligrams of (6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine in a once daily modified release pharmaceutical composition wherein said patient is a naive patient and wherein the modified release composition releases the (6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine over a 24 hour period.
 8. The method of claim 7, wherein said neurodegenerative disease is amyotrophic lateral sclerosis.
 9. The method of claim 7, wherein the chiral purity for the (6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine is 99.9%, or greater.
 10. The method of claim 7, wherein the chiral purity for the (6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine is 99.95%, or greater.
 11. The method of claim 7, wherein the chiral purity for the (6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine is 99.99% or greater. 